Methods and compositions for diagnosis and prognosis of renal injury and renal failure

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using a one or more assays configured to detect a kidney injury marker selected from the group consisting of Coagulation factor VII, CA19-9, Insulin-like growth factor-binding protein 7, C—X—C motif chemokine 6, and C—C motif chemokine 13 as diagnostic and prognostic biomarkers in renal injuries.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is filed under 35 U.S.C. § 371 as the U.S.national phase of International Application No. PCT/US2011/023830, filedFeb. 4, 2011, which designated the U.S. and claims the benefit ofpriority to U.S. Provisional Patent Application 61/302,021 filed Feb. 5,2010; U.S. Provisional Patent Application No. 61/302,025 filed Feb. 5,2010; U.S. Provisional Patent Application No. 61/302,026 filed Feb. 5,2010; U.S. Provisional Patent Application No. 61/302,027 filed Feb. 5,2010; U.S. Provisional Patent Application No. 61/302,036 filed Feb. 5,2010; and U.S. Provisional Patent Application No. 61/374,060 filed Aug.16, 2010, each of which is hereby incorporated in its entirety includingall tables, figures, and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 4, 2012, isnamed AST1610US_SeqList_txt.txt and is 8,958 bytes in size.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

The kidney is responsible for water and solute excretion from the body.Its functions include maintenance of acid-base balance, regulation ofelectrolyte concentrations, control of blood volume, and regulation ofblood pressure. As such, loss of kidney function through injury and/ordisease results in substantial morbidity and mortality. A detaileddiscussion of renal injuries is provided in Harrison's Principles ofInternal Medicine, 17^(th) Ed., McGraw Hill, New York, pages 1741-1830,which are hereby incorporated by reference in their entirety. Renaldisease and/or injury may be acute or chronic. Acute and chronic kidneydisease are described as follows (from Current Medical Diagnosis &Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, whichare hereby incorporated by reference in their entirety): “Acute renalfailure is worsening of renal function over hours to days, resulting inthe retention of nitrogenous wastes (such as urea nitrogen) andcreatinine in the blood. Retention of these substances is calledazotemia. Chronic renal failure (chronic kidney disease) results from anabnormal loss of renal function over months to years”.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) isan abrupt (typically detected within about 48 hours to 1 week)reductionin glomerular filtration. This loss of filtration capacity results inretention of nitrogenous (urea and creatinine) and non-nitrogenous wasteproducts that are normally excreted by the kidney, a reduction in urineoutput, or both. It is reported that ARF complicates about 5% ofhospital admissions, 4-15% of cardiopulmonary bypass surgeries, and upto 30% of intensive care admissions. ARF may be categorized as prerenal,intrinsic renal, or postrenal in causation. Intrinsic renal disease canbe further divided into glomerular, tubular, interstitial, and vascularabnormalities. Major causes of ARF are described in the following table,which is adapted from the Merck Manual, 17^(th) ed., Chapter 222, andwhich is hereby incorporated by reference in their entirety:

Type Risk Factors Prerenal ECF volume depletion Excessive diuresis,hemorrhage, GI losses, loss of intravascular fluid into theextravascular space (due to ascites, peritonitis, pancreatitis, orburns), loss of skin and mucus membranes, renal salt- and water-wastingstates Low cardiac output Cardiomyopathy, MI, cardiac tamponade,pulmonary embolism, pulmonary hypertension, positive-pressure mechanicalventilation Low systemic vascular Septic shock, liver failure,antihypertensive drugs resistance Increased renal vascular NSAIDs,cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis,anesthetics, renal artery obstruction, renal vein thrombosis, sepsis,hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin IIreceptor blockers arteriolar tone (leading to decreased GFR from reducedglomerular transcapillary pressure, especially in patients withbilateral renal artery stenosis) Intrinsic Renal Acute tubular injuryIschemia (prolonged or severe prerenal state): surgery, hemorrhage,arterial or venous obstruction; Toxins: NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, streptozotocin Acute glomerulonephritis ANCA-associated:Crescentic glomerulonephritis, polyarteritis nodosa, Wegener'sgranulomatosis; Anti- GBM glomerulonephritis: Goodpasture's syndrome;Immune-complex: Lupus glomerulonephritis, postinfectiousglomerulonephritis, cryoglobulinemic glomerulonephritis Acutetubulointerstitial Drug reaction (eg, β-lactams, NSAIDs, sulfonamides,nephritis ciprofloxacin, thiazide diuretics, furosemide, phenytoin,allopurinol, pyelonephritis, papillary necrosis Acute vascularVasculitis, malignant hypertension, thrombotic nephropathymicroangiopathies, scleroderma, atheroembolism Infiltrative diseasesLymphoma, sarcoidosis, leukemia Postrenal Tubular precipitation Uricacid (tumor lysis), sulfonamides, triamterene, acyclovir, indinavir,methotrexate, ethylene glycol ingestion, myeloma protein, myoglobinUreteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue,fungus ball, edema, malignancy, congenital defects; Extrinsic:Malignancy, retroperitoneal fibrosis, ureteral trauma during surgery orhigh impact injury Bladder obstruction Mechanical: Benign prostatichyperplasia, prostate cancer, bladder cancer, urethral strictures,phimosis, paraphimosis, urethral valves, obstructed indwelling urinarycatheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuronlesion

In the case of ischemic ARF, the course of the disease may be dividedinto four phases. During an initiation phase, which lasts hours to days,reduced perfusion of the kidney is evolving into injury. Glomerularultrafiltration reduces, the flow of filtrate is reduced due to debriswithin the tubules, and back leakage of filtrate through injuredepithelium occurs. Renal injury can be mediated during this phase byreperfusion of the kidney. Initiation is followed by an extension phasewhich is characterized by continued ischemic injury and inflammation andmay involve endothelial damage and vascular congestion. During themaintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs,and glomerular filtration and urine output reaches a minimum. A recoveryphase can follow in which the renal epithelium is repaired and GFRgradually recovers. Despite this, the survival rate of subjects with ARFmay be as low as about 60%.

Acute kidney injury caused by radiocontrast agents (also called contrastmedia) and other nephrotoxins such as cyclosporine, antibioticsincluding aminoglycosides and anticancer drugs such as cisplatinmanifests over a period of days to about a week. Contrast inducednephropathy (CIN, which is AKI caused by radiocontrast agents) isthought to be caused by intrarenal vasoconstriction (leading to ischemicinjury) and from the generation of reactive oxygen species that aredirectly toxic to renal tubular epithelial cells. CIN classicallypresents as an acute (onset within 24-48h) but reversible (peak 3-5days, resolution within 1 week) rise in blood urea nitrogen and serumcreatinine.

A commonly reported criteria for defining and detecting AKI is an abrupt(typically within about 2-7 days or within a period of hospitalization)elevation of serum creatinine. Although the use of serum creatinineelevation to define and detect AKI is well established, the magnitude ofthe serum creatinine elevation and the time over which it is measured todefine AKI varies considerably among publications. Traditionally,relatively large increases in serum creatinine such as 100%, 200%, anincrease of at least 100% to a value over 2 mg/dL and other definitionswere used to define AKI. However, the recent trend has been towardsusing smaller serum creatinine rises to define AKI. The relationshipbetween serum creatinine rise, AKI and the associated health risks arereviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270,2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which,with the references listed therein, are hereby incorporated by referencein their entirety. As described in these publications, acute worseningrenal function (AKI) and increased risk of death and other detrimentaloutcomes are now known to be associated with very small increases inserum creatinine. These increases may be determined as a relative(percent) value or a nominal value. Relative increases in serumcreatinine as small as 20% from the pre-injury value have been reportedto indicate acutely worsening renal function (AKI) and increased healthrisk, but the more commonly reported value to define AKI and increasedhealth risk is a relative increase of at least 25%. Nominal increases assmall as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported toindicate worsening renal function and increased risk of death. Varioustime periods for the serum creatinine to rise to these threshold valueshave been used to define AKI, for example, ranging from 2 days, 3 days,7 days, or a variable period defined as the time the patient is in thehospital or intensive care unit. These studies indicate there is not aparticular threshold serum creatinine rise (or time period for the rise)for worsening renal function or AKI, but rather a continuous increase inrisk with increasing magnitude of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, herebyincorporated by reference in its entirety) investigated both increasesand decreases in serum creatinine. Patients with a mild fall in serumcreatinine of −0.1 to −0.3 mg/dL following heart surgery had the lowestmortality rate. Patients with a larger fall in serum creatinine (morethan or equal to −0.4 mg/dL) or any increase in serum creatinine had alarger mortality rate. These findings caused the authors to concludethat even very subtle changes in renal function (as detected by smallcreatinine changes within 48 hours of surgery) seriously effectpatient's outcomes. In an effort to reach consensus on a unifiedclassification system for using serum creatinine to define AKI inclinical trials and in clinical practice, Bellomo et al., Crit. Care.8(4):R204-12, 2004, which is hereby incorporated by reference in itsentirety, proposes the following classifications for stratifying AKIpatients:

“Risk”: serum creatinine increased 1.5 fold from baseline OR urineproduction of <0.5 ml/kg body weight/hr for 6 hours;

“Injury”: serum creatinine increased 2.0 fold from baseline OR urineproduction <0.5 ml/kg/hr for 12 h;

“Failure”: serum creatinine increased 3.0 fold from baseline ORcreatinine >355 μmol/l (with a rise of >44) or urine output below 0.3ml/kg/hr for 24 h or anuria for at least 12 hours;

And included two clinical outcomes:

“Loss”: persistent need for renal replacement therapy for more than fourweeks.

“ESRD”: end stage renal disease—the need for dialysis for more than 3months.

These criteria are called the RIFLE criteria, which provide a usefulclinical tool to classify renal status. As discussed in Kellum, Crit.Care Med. 36: S141-45, 2008 and Ricci et al., Kidney Int. 73, 538-546,2008, each hereby incorporated by reference in its entirety, the RIFLEcriteria provide a uniform definition of AKI which has been validated innumerous studies.

More recently, Mehta et al., Crit. Care 11:R31 (doi:10.1186.cc5713),2007, hereby incorporated by reference in its entirety, proposes thefollowing similar classifications for stratifying AKI patients, whichhave been modified from RIFLE:

“Stage I”: increase in serum creatinine of more than or equal to 0.3mg/dL (≥26.4 μmol/L) or increase to more than or equal to 150%(1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hourfor more than 6 hours;

“Stage II”: increase in serum creatinine to more than 200% (>2-fold)from baseline OR urine output less than 0.5 mL/kg per hour for more than12 hours;

“Stage III”: increase in serum creatinine to more than 300% (>3-fold)from baseline OR serum creatinine ≥354 μmol/L accompanied by an acuteincrease of at least 44 μmol/L OR urine output less than 0.3 mL/kg perhour for 24 hours or anuria for 12 hours.

The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med.2006; 7(4): 177-197, hereby incorporated by reference in its entirety)uses a serum creatinine rise of 25% to define Contrast inducednephropathy (which is a type of AKI).Although various groups proposeslightly different criteria for using serum creatinine to detect AKI,the consensus is that small changes in serum creatinine, such as 0.3mg/dL or 25%, are sufficient to detect AKI (worsening renal function)and that the magnitude of the serum creatinine change is an indicator ofthe severity of the AKI and mortality risk.

Although serial measurement of serum creatinine over a period of days isan accepted method of detecting and diagnosing AKI and is considered oneof the most important tools to evaluate AKI patients, serum creatinineis generally regarded to have several limitations in the diagnosis,assessment and monitoring of AKI patients. The time period for serumcreatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considereddiagnostic for AKI can be 48 hours or longer depending on the definitionused. Since cellular injury in AKI can occur over a period of hours,serum creatinine elevations detected at 48 hours or longer can be a lateindicator of injury, and relying on serum creatinine can thus delaydiagnosis of AKI. Furthermore, serum creatinine is not a good indicatorof the exact kidney status and treatment needs during the most acutephases of AKI when kidney function is changing rapidly. Some patientswith AKI will recover fully, some will need dialysis (either short termor long term) and some will have other detrimental outcomes includingdeath, major adverse cardiac events and chronic kidney disease. Becauseserum creatinine is a marker of filtration rate, it does notdifferentiate between the causes of AKI (pre-renal, intrinsic renal,post-renal obstruction, atheroembolic, etc) or the category or locationof injury in intrinsic renal disease (for example, tubular, glomerularor interstitial in origin). Urine output is similarly limited, Knowingthese things can be of vital importance in managing and treatingpatients with AKI.

These limitations underscore the need for better methods to detect andassess AKI, particularly in the early and subclinical stages, but alsoin later stages when recovery and repair of the kidney can occur.Furthermore, there is a need to better identify patients who are at riskof having an AKI.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions forevaluating renal function in a subject. As described herein, measurementof one or more biomarkers selected from the group consisting ofCoagulation factor VII, CA19-9, Insulin-like growth factor-bindingprotein 7, C—X—C motif chemokine 6, and C—C motif chemokine 13 (eachreferred to herein as a “kidney injury marker”) can be used fordiagnosis, prognosis, risk stratification, staging, monitoring,categorizing and determination of further diagnosis and treatmentregimens in subjects suffering or at risk of suffering from an injury torenal function, reduced renal function, and/or acute renal failure (alsocalled acute kidney injury).

The kidney injury markers of the present invention may be used,individually or in panels comprising a plurality of kidney injurymarkers, for risk stratification (that is, to identify subjects at riskfor a future injury to renal function, for future progression to reducedrenal function, for future progression to ARF, for future improvement inrenal function, etc.); for diagnosis of existing disease (that is, toidentify subjects who have suffered an injury to renal function, whohave progressed to reduced renal function, who have progressed to ARF,etc.); for monitoring for deterioration or improvement of renalfunction; and for predicting a future medical outcome, such as improvedor worsening renal function, a decreased or increased mortality risk, adecreased or increased risk that a subject will require renalreplacement therapy (i.e., hemodialysis, peritoneal dialysis,hemofiltration, and/or renal transplantation, a decreased or increasedrisk that a subject will recover from an injury to renal function, adecreased or increased risk that a subject will recover from ARF, adecreased or increased risk that a subject will progress to end stagerenal disease, a decreased or increased risk that a subject willprogress to chronic renal failure, a decreased or increased risk that asubject will suffer rejection of a transplanted kidney, etc.

In a first aspect, the present invention relates to methods forevaluating renal status in a subject. These methods comprise performingan assay method that is configured to detect one or more biomarkersselected from the group consisting of Coagulation factor VII, CA19-9,Insulin-like growth factor-binding protein 7, C—X—C motif chemokine 6,and C—C motif chemokine 13 is/are then correlated to the renal status ofthe subject. This correlation to renal status may include correlatingthe assay result(s) to one or more of risk stratification, diagnosis,prognosis, staging, classifying and monitoring of the subject asdescribed herein. Thus, the present invention utilizes one or morekidney injury markers of the present invention for the evaluation ofrenal injury.

In certain embodiments, the methods for evaluating renal statusdescribed herein are methods for risk stratification of the subject;that is, assigning a likelihood of one or more future changes in renalstatus to the subject. In these embodiments, the assay result(s) is/arecorrelated to one or more such future changes. The following arepreferred risk stratification embodiments.

In preferred risk stratification embodiments, these methods comprisedetermining a subject's risk for a future injury to renal function, andthe assay result(s) is/are correlated to a likelihood of such a futureinjury to renal function. For example, the measured concentration(s) mayeach be compared to a threshold value. For a “positive going” kidneyinjury marker, an increased likelihood of suffering a future injury torenal function is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of suffering afuture injury to renal function is assigned to the subject when themeasured concentration is below the threshold, relative to a likelihoodassigned when the measured concentration is above the threshold.

In other preferred risk stratification embodiments, these methodscomprise determining a subject's risk for future reduced renal function,and the assay result(s) is/are correlated to a likelihood of suchreduced renal function. For example, the measured concentrations mayeach be compared to a threshold value. For a “positive going” kidneyinjury marker, an increased likelihood of suffering a future reducedrenal function is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of future reducedrenal function is assigned to the subject when the measuredconcentration is below the threshold, relative to a likelihood assignedwhen the measured concentration is above the threshold.

In still other preferred risk stratification embodiments, these methodscomprise determining a subject's likelihood for a future improvement inrenal function, and the assay result(s) is/are correlated to alikelihood of such a future improvement in renal function. For example,the measured concentration(s) may each be compared to a threshold value.For a “positive going” kidney injury marker, an increased likelihood ofa future improvement in renal function is assigned to the subject whenthe measured concentration is below the threshold, relative to alikelihood assigned when the measured concentration is above thethreshold. For a “negative going” kidney injury marker, an increasedlikelihood of a future improvement in renal function is assigned to thesubject when the measured concentration is above the threshold, relativeto a likelihood assigned when the measured concentration is below thethreshold.

In yet other preferred risk stratification embodiments, these methodscomprise determining a subject's risk for progression to ARF, and theresult(s) is/are correlated to a likelihood of such progression to ARF.For example, the measured concentration(s) may each be compared to athreshold value. For a “positive going” kidney injury marker, anincreased likelihood of progression to ARF is assigned to the subjectwhen the measured concentration is above the threshold, relative to alikelihood assigned when the measured concentration is below thethreshold. For a “negative going” kidney injury marker, an increasedlikelihood of progression to ARF is assigned to the subject when themeasured concentration is below the threshold, relative to a likelihoodassigned when the measured concentration is above the threshold.

And in other preferred risk stratification embodiments, these methodscomprise determining a subject's outcome risk, and the assay result(s)is/are correlated to a likelihood of the occurrence of a clinicaloutcome related to a renal injury suffered by the subject. For example,the measured concentration(s) may each be compared to a threshold value.For a “positive going” kidney injury marker, an increased likelihood ofone or more of: acute kidney injury, progression to a worsening stage ofAKI, mortality, a requirement for renal replacement therapy, arequirement for withdrawal of renal toxins, end stage renal disease,heart failure, stroke, myocardial infarction, progression to chronickidney disease, etc., is assigned to the subject when the measuredconcentration is above the threshold, relative to a likelihood assignedwhen the measured concentration is below the threshold. For a “negativegoing” kidney injury marker, an increased likelihood of one or more of:acute kidney injury, progression to a worsening stage of AKI, mortality,a requirement for renal replacement therapy, a requirement forwithdrawal of renal toxins, end stage renal disease, heart failure,stroke, myocardial infarction, progression to chronic kidney disease,etc., is assigned to the subject when the measured concentration isbelow the threshold, relative to a likelihood assigned when the measuredconcentration is above the threshold.

In such risk stratification embodiments, preferably the likelihood orrisk assigned is that an event of interest is more or less likely tooccur within 180 days of the time at which the body fluid sample isobtained from the subject. In particularly preferred embodiments, thelikelihood or risk assigned relates to an event of interest occurringwithin a shorter time period such as 18 months, 120 days, 90 days, 60days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0hours of the time at which the body fluid sample is obtained from thesubject is equivalent to diagnosis of a current condition.

In preferred risk stratification embodiments, the subject is selectedfor risk stratification based on the pre-existence in the subject of oneor more known risk factors for prerenal, intrinsic renal, or postrenalARF. For example, a subject undergoing or having undergone majorvascular surgery, coronary artery bypass, or other cardiac surgery; asubject having pre-existing congestive heart failure, preeclampsia,eclampsia, diabetes mellitus, hypertension, coronary artery disease,proteinuria, renal insufficiency, glomerular filtration below the normalrange, cirrhosis, serum creatinine above the normal range, or sepsis; ora subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides,foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavymetals, methotrexate, radiopaque contrast agents, or streptozotocin areall preferred subjects for monitoring risks according to the methodsdescribed herein. This list is not meant to be limiting. By“pre-existence” in this context is meant that the risk factor exists atthe time the body fluid sample is obtained from the subject. Inparticularly preferred embodiments, a subject is chosen for riskstratification based on an existing diagnosis of injury to renalfunction, reduced renal function, or ARF.

In other embodiments, the methods for evaluating renal status describedherein are methods for diagnosing a renal injury in the subject; thatis, assessing whether or not a subject has suffered from an injury torenal function, reduced renal function, or ARF. In these embodiments,the assay result(s), for example measured concentration(s) of one ormore biomarkers selected from the group consisting of Coagulation factorVII, CA19-9, Insulin-like growth factor-binding protein 7, C—X—C motifchemokine 6, and C—C motif chemokine 13 is/are correlated to theoccurrence or nonoccurrence of a change in renal status. The followingare preferred diagnostic embodiments.

In preferred diagnostic embodiments, these methods comprise diagnosingthe occurrence or nonoccurrence of an injury to renal function, and theassay result(s) is/are correlated to the occurrence or nonoccurrence ofsuch an injury. For example, each of the measured concentration(s) maybe compared to a threshold value. For a positive going marker, anincreased likelihood of the occurrence of an injury to renal function isassigned to the subject when the measured concentration is above thethreshold (relative to the likelihood assigned when the measuredconcentration is below the threshold); alternatively, when the measuredconcentration is below the threshold, an increased likelihood of thenonoccurrence of an injury to renal function may be assigned to thesubject (relative to the likelihood assigned when the measuredconcentration is above the threshold). For a negative going marker, anincreased likelihood of the occurrence of an injury to renal function isassigned to the subject when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of an injury to renal function may be assigned to thesubject (relative to the likelihood assigned when the measuredconcentration is below the threshold).

In other preferred diagnostic embodiments, these methods comprisediagnosing the occurrence or nonoccurrence of reduced renal function,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of an injury causing reduced renal function. For example,each of the measured concentration(s) may be compared to a thresholdvalue. For a positive going marker, an increased likelihood of theoccurrence of an injury causing reduced renal function is assigned tothe subject when the measured concentration is above the threshold(relative to the likelihood assigned when the measured concentration isbelow the threshold); alternatively, when the measured concentration isbelow the threshold, an increased likelihood of the nonoccurrence of aninjury causing reduced renal function may be assigned to the subject(relative to the likelihood assigned when the measured concentration isabove the threshold). For a negative going marker, an increasedlikelihood of the occurrence of an injury causing reduced renal functionis assigned to the subject when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of an injury causing reduced renal function may beassigned to the subject (relative to the likelihood assigned when themeasured concentration is below the threshold).

In yet other preferred diagnostic embodiments, these methods comprisediagnosing the occurrence or nonoccurrence of ARF, and the assayresult(s) is/are correlated to the occurrence or nonoccurrence of aninjury causing ARF. For example, each of the measured concentration(s)may be compared to a threshold value. For a positive going marker, anincreased likelihood of the occurrence of ARF is assigned to the subjectwhen the measured concentration is above the threshold (relative to thelikelihood assigned when the measured concentration is below thethreshold); alternatively, when the measured concentration is below thethreshold, an increased likelihood of the nonoccurrence of ARF may beassigned to the subject (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of ARF is assigned tothe subject when the measured concentration is below the threshold(relative to the likelihood assigned when the measured concentration isabove the threshold); alternatively, when the measured concentration isabove the threshold, an increased likelihood of the nonoccurrence of ARFmay be assigned to the subject (relative to the likelihood assigned whenthe measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprisediagnosing a subject as being in need of renal replacement therapy, andthe assay result(s) is/are correlated to a need for renal replacementtherapy. For example, each of the measured concentration(s) may becompared to a threshold value. For a positive going marker, an increasedlikelihood of the occurrence of an injury creating a need for renalreplacement therapy is assigned to the subject when the measuredconcentration is above the threshold (relative to the likelihoodassigned when the measured concentration is below the threshold);alternatively, when the measured concentration is below the threshold,an increased likelihood of the nonoccurrence of an injury creating aneed for renal replacement therapy may be assigned to the subject(relative to the likelihood assigned when the measured concentration isabove the threshold). For a negative going marker, an increasedlikelihood of the occurrence of an injury creating a need for renalreplacement therapy is assigned to the subject when the measuredconcentration is below the threshold (relative to the likelihoodassigned when the measured concentration is above the threshold);alternatively, when the measured concentration is above the threshold,an increased likelihood of the nonoccurrence of an injury creating aneed for renal replacement therapy may be assigned to the subject(relative to the likelihood assigned when the measured concentration isbelow the threshold).

In still other preferred diagnostic embodiments, these methods comprisediagnosing a subject as being in need of renal transplantation, and theassay result (s0 is/are correlated to a need for renal transplantation.For example, each of the measured concentration(s) may be compared to athreshold value. For a positive going marker, an increased likelihood ofthe occurrence of an injury creating a need for renal transplantation isassigned to the subject when the measured concentration is above thethreshold (relative to the likelihood assigned when the measuredconcentration is below the threshold); alternatively, when the measuredconcentration is below the threshold, an increased likelihood of thenonoccurrence of an injury creating a need for renal transplantation maybe assigned to the subject (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of an injury creatinga need for renal transplantation is assigned to the subject when themeasured concentration is below the threshold (relative to thelikelihood assigned when the measured concentration is above thethreshold); alternatively, when the measured concentration is above thethreshold, an increased likelihood of the nonoccurrence of an injurycreating a need for renal transplantation may be assigned to the subject(relative to the likelihood assigned when the measured concentration isbelow the threshold).

In still other embodiments, the methods for evaluating renal statusdescribed herein are methods for monitoring a renal injury in thesubject; that is, assessing whether or not renal function is improvingor worsening in a subject who has suffered from an injury to renalfunction, reduced renal function, or ARF. In these embodiments, theassay result(s), for example measured concentration(s) of one or morebiomarkers selected from the group consisting of Coagulation factor VII,CA19-9, Insulin-like growth factor-binding protein 7, C—X—C motifchemokine 6, and C—C motif chemokine 13 is/are correlated to theoccurrence or nonoccurrence of a change in renal status. The followingare preferred monitoring embodiments.

In preferred monitoring embodiments, these methods comprise monitoringrenal status in a subject suffering from an injury to renal function,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In other preferred monitoring embodiments, these methods comprisemonitoring renal status in a subject suffering from reduced renalfunction, and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprisemonitoring renal status in a subject suffering from acute renal failure,and the assay result(s) is/are correlated to the occurrence ornonoccurrence of a change in renal status in the subject. For example,the measured concentration(s) may be compared to a threshold value. Fora positive going marker, when the measured concentration is above thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In other additional preferred monitoring embodiments, these methodscomprise monitoring renal status in a subject at risk of an injury torenal function due to the pre-existence of one or more known riskfactors for prerenal, intrinsic renal, or postrenal ARF, and the assayresult(s) is/are correlated to the occurrence or nonoccurrence of achange in renal status in the subject. For example, the measuredconcentration(s) may be compared to a threshold value. For a positivegoing marker, when the measured concentration is above the threshold, aworsening of renal function may be assigned to the subject;alternatively, when the measured concentration is below the threshold,an improvement of renal function may be assigned to the subject. For anegative going marker, when the measured concentration is below thethreshold, a worsening of renal function may be assigned to the subject;alternatively, when the measured concentration is above the threshold,an improvement of renal function may be assigned to the subject.

In still other embodiments, the methods for evaluating renal statusdescribed herein are methods for classifying a renal injury in thesubject; that is, determining whether a renal injury in a subject isprerenal, intrinsic renal, or postrenal; and/or further subdividingthese classes into subclasses such as acute tubular injury, acuteglomerulonephritis acute tubulointerstitial nephritis, acute vascularnephropathy, or infiltrative disease; and/or assigning a likelihood thata subject will progress to a particular RIFLE stage. In theseembodiments, the assay result(s), for example measured concentration(s)of one or more biomarkers selected from the group consisting ofCoagulation factor VII, CA19-9, Insulin-like growth factor-bindingprotein 7, C—X—C motif chemokine 6, and C—C motif chemokine 13 is/arecorrelated to a particular class and/or subclass. The following arepreferred classification embodiments.

In preferred classification embodiments, these methods comprisedetermining whether a renal injury in a subject is prerenal, intrinsicrenal, or postrenal; and/or further subdividing these classes intosubclasses such as acute tubular injury, acute glomerulonephritis acutetubulointerstitial nephritis, acute vascular nephropathy, orinfiltrative disease; and/or assigning a likelihood that a subject willprogress to a particular RIFLE stage, and the assay result(s) is/arecorrelated to the injury classification for the subject. For example,the measured concentration may be compared to a threshold value, andwhen the measured concentration is above the threshold, a particularclassification is assigned; alternatively, when the measuredconcentration is below the threshold, a different classification may beassigned to the subject.

A variety of methods may be used by the skilled artisan to arrive at adesired threshold value for use in these methods. For example, thethreshold value may be determined from a population of normal subjectsby selecting a concentration representing the 75^(th), 85^(th), 90^(th),95^(th), or 99^(th) percentile of a kidney injury marker measured insuch normal subjects. Alternatively, the threshold value may bedetermined from a “diseased” population of subjects, e.g., thosesuffering from an injury or having a predisposition for an injury (e.g.,progression to ARF or some other clinical outcome such as death,dialysis, renal transplantation, etc.), by selecting a concentrationrepresenting the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th)percentile of a kidney injury marker measured in such subjects. Inanother alternative, the threshold value may be determined from a priormeasurement of a kidney injury marker in the same subject; that is, atemporal change in the level of a kidney injury marker in the subjectmay be used to assign risk to the subject.

The foregoing discussion is not meant to imply, however, that the kidneyinjury markers of the present invention must be compared tocorresponding individual thresholds. Methods for combining assay resultscan comprise the use of multivariate logistical regression, loglinearmodeling, neural network analysis, n-of-m analysis, decision treeanalysis, calculating ratios of markers, etc. This list is not meant tobe limiting. In these methods, a composite result which is determined bycombining individual markers may be treated as if it is itself a marker;that is, a threshold may be determined for the composite result asdescribed herein for individual markers, and the composite result for anindividual patient compared to this threshold.

The ability of a particular test to distinguish two populations can beestablished using ROC analysis. For example, ROC curves established froma “first” subpopulation which is predisposed to one or more futurechanges in renal status, and a “second” subpopulation which is not sopredisposed can be used to calculate a ROC curve, and the area under thecurve provides a measure of the quality of the test. Preferably, thetests described herein provide a ROC curve area greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95.

In certain aspects, the measured concentration of one or more kidneyinjury markers, or a composite of such markers, may be treated ascontinuous variables. For example, any particular concentration can beconverted into a corresponding probability of a future reduction inrenal function for the subject, the occurrence of an injury, aclassification, etc. In yet another alternative, a threshold that canprovide an acceptable level of specificity and sensitivity in separatinga population of subjects into “bins” such as a “first” subpopulation(e.g., which is predisposed to one or more future changes in renalstatus, the occurrence of an injury, a classification, etc.) and a“second” subpopulation which is not so predisposed. A threshold value isselected to separate this first and second population by one or more ofthe following measures of test accuracy:

an odds ratio greater than 1, preferably at least about 2 or more orabout 0.5 or less, more preferably at least about 3 or more or about0.33 or less, still more preferably at least about 4 or more or about0.25 or less, even more preferably at least about 5 or more or about 0.2or less, and most preferably at least about 10 or more or about 0.1 orless;a specificity of greater than 0.5, preferably at least about 0.6, morepreferably at least about 0.7, still more preferably at least about 0.8,even more preferably at least about 0.9 and most preferably at leastabout 0.95, with a corresponding sensitivity greater than 0.2,preferably greater than about 0.3, more preferably greater than about0.4, still more preferably at least about 0.5, even more preferablyabout 0.6, yet more preferably greater than about 0.7, still morepreferably greater than about 0.8, more preferably greater than about0.9, and most preferably greater than about 0.95;a sensitivity of greater than 0.5, preferably at least about 0.6, morepreferably at least about 0.7, still more preferably at least about 0.8,even more preferably at least about 0.9 and most preferably at leastabout 0.95, with a corresponding specificity greater than 0.2,preferably greater than about 0.3, more preferably greater than about0.4, still more preferably at least about 0.5, even more preferablyabout 0.6, yet more preferably greater than about 0.7, still morepreferably greater than about 0.8, more preferably greater than about0.9, and most preferably greater than about 0.95;at least about 75% sensitivity, combined with at least about 75%specificity;a positive likelihood ratio (calculated as sensitivity/(1-specificity))of greater than 1, at least about 2, more preferably at least about 3,still more preferably at least about 5, and most preferably at leastabout 10; ora negative likelihood ratio (calculated as (1-sensitivity)/specificity)of less than 1, less than or equal to about 0.5, more preferably lessthan or equal to about 0.3, and most preferably less than or equal toabout 0.1.

The term “about” in the context of any of the above measurements refersto +/−5% of a given measurement.

Multiple thresholds may also be used to assess renal status in asubject. For example, a “first” subpopulation which is predisposed toone or more future changes in renal status, the occurrence of an injury,a classification, etc., and a “second” subpopulation which is not sopredisposed can be combined into a single group. This group is thensubdivided into three or more equal parts (known as tertiles, quartiles,quintiles, etc., depending on the number of subdivisions). An odds ratiois assigned to subjects based on which subdivision they fall into. Ifone considers a tertile, the lowest or highest tertile can be used as areference for comparison of the other subdivisions. This referencesubdivision is assigned an odds ratio of 1. The second tertile isassigned an odds ratio that is relative to that first tertile. That is,someone in the second tertile might be 3 times more likely to suffer oneor more future changes in renal status in comparison to someone in thefirst tertile. The third tertile is also assigned an odds ratio that isrelative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodiesfor use in such assays will specifically bind a full length kidneyinjury marker of interest, and may also bind one or more polypeptidesthat are “related” thereto, as that term is defined hereinafter.Numerous immunoassay formats are known to those of skill in the art.Preferred body fluid samples are selected from the group consisting ofurine, blood, serum, saliva, tears, and plasma.

The foregoing method steps should not be interpreted to mean that thekidney injury marker assay result(s) is/are used in isolation in themethods described herein. Rather, additional variables or other clinicalindicia may be included in the methods described herein. For example, arisk stratification, diagnostic, classification, monitoring, etc. methodmay combine the assay result(s) with one or more variables measured forthe subject selected from the group consisting of demographicinformation (e.g., weight, sex, age, race), medical history (e.g.,family history, type of surgery, pre-existing disease such as aneurism,congestive heart failure, preeclampsia, eclampsia, diabetes mellitus,hypertension, coronary artery disease, proteinuria, renal insufficiency,or sepsis, type of toxin exposure such as NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, or streptozotocin), clinical variables (e.g., blood pressure,temperature, respiration rate), risk scores (APACHE score, PREDICTscore, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score, risk scoresof Thakar et al. (J. Am. Soc. Nephrol. 16: 162-68, 2005), Mehran et al.(J. Am. Coll. Cardiol. 44: 1393-99, 2004), Wijeysundera et al. (JAMA297: 1801-9, 2007), Goldstein and Chawla (Clin. J. Am. Soc. Nephrol. 5:943-49, 2010), or Chawla et al. (Kidney Intl. 68: 2274-80, 2005)), aglomerular filtration rate, an estimated glomerular filtration rate, aurine production rate, a serum or plasma creatinine concentration, aurine creatinine concentration, a fractional excretion of sodium, aurine sodium concentration, a urine creatinine to serum or plasmacreatinine ratio, a urine specific gravity, a urine osmolality, a urineurea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnineratio, a renal failure index calculated as urine sodium/(urinecreatinine/plasma creatinine), a serum or plasma neutrophil gelatinase(NGAL) concentration, a urine NGAL concentration, a serum or plasmacystatin C concentration, a serum or plasma cardiac troponinconcentration, a serum or plasma BNP concentration, a serum or plasmaNTproBNP concentration, and a serum or plasma proBNP concentration.Other measures of renal function which may be combined with one or morekidney injury marker assay result(s) are described hereinafter and inHarrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill,New York, pages 1741-1830, and Current Medical Diagnosis & Treatment2008, 47^(th) Ed, McGraw Hill, New York, pages 785-815, each of whichare hereby incorporated by reference in their entirety.

When more than one marker is measured, the individual markers may bemeasured in samples obtained at the same time, or may be determined fromsamples obtained at different (e.g., an earlier or later) times. Theindividual markers may also be measured on the same or different bodyfluid samples. For example, one kidney injury marker may be measured ina serum or plasma sample and another kidney injury marker may bemeasured in a urine sample. In addition, assignment of a likelihood maycombine an individual kidney injury marker assay result with temporalchanges in one or more additional variables.

In various related aspects, the present invention also relates todevices and kits for performing the methods described herein. Suitablekits comprise reagents sufficient for performing an assay for at leastone of the described kidney injury markers, together with instructionsfor performing the described threshold comparisons.

In certain embodiments, reagents for performing such assays are providedin an assay device, and such assay devices may be included in such akit. Preferred reagents can comprise one or more solid phase antibodies,the solid phase antibody comprising antibody that detects the intendedbiomarker target(s) bound to a solid support. In the case of sandwichimmunoassays, such reagents can also include one or more detectablylabeled antibodies, the detectably labeled antibody comprising antibodythat detects the intended biomarker target(s) bound to a detectablelabel. Additional optional elements that may be provided as part of anassay device are described hereinafter.

Detectable labels may include molecules that are themselves detectable(e.g., fluorescent moieties, electrochemical labels, ecl(electrochemical luminescence) labels, metal chelates, colloidal metalparticles, etc.) as well as molecules that may be indirectly detected byproduction of a detectable reaction product (e.g., enzymes such ashorseradish peroxidase, alkaline phosphatase, etc.) or through the useof a specific binding molecule which itself may be detectable (e.g., alabeled antibody that binds to the second antibody, biotin, digoxigenin,maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA,dsDNA, etc.).

Generation of a signal from the signal development element can beperformed using various optical, acoustical, and electrochemical methodswell known in the art. Examples of detection modes include fluorescence,radiochemical detection, reflectance, absorbance, amperometry,conductance, impedance, interferometry, ellipsometry, etc. In certain ofthese methods, the solid phase antibody is coupled to a transducer(e.g., a diffraction grating, electrochemical sensor, etc) forgeneration of a signal, while in others, a signal is generated by atransducer that is spatially separate from the solid phase antibody(e.g., a fluorometer that employs an excitation light source and anoptical detector). This list is not meant to be limiting. Antibody-basedbiosensors may also be employed to determine the presence or amount ofanalytes that optionally eliminate the need for a labeled molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis,differential diagnosis, risk stratification, monitoring, classifying anddetermination of treatment regimens in subjects suffering or at risk ofsuffering from injury to renal function, reduced renal function and/oracute renal failure through measurement of one or more kidney injurymarkers. In various embodiments, a measured concentration of one or morebiomarkers selected from the group consisting of Coagulation factor VII,CA19-9, Insulin-like growth factor-binding protein 7, C—X—C motifchemokine 6, and C—C motif chemokine 13 or one or more markers relatedthereto, are correlated to the renal status of the subject.

For purposes of this document, the following definitions apply:

As used herein, an “injury to renal function” is an abrupt (within 14days, preferably within 7 days, more preferably within 72 hours, andstill more preferably within 48 hours) measurable reduction in a measureof renal function. Such an injury may be identified, for example, by adecrease in glomerular filtration rate or estimated GFR, a reduction inurine output, an increase in serum creatinine, an increase in serumcystatin C, a requirement for renal replacement therapy, etc.“Improvement in Renal Function” is an abrupt (within 14 days, preferablywithin 7 days, more preferably within 72 hours, and still morepreferably within 48 hours) measurable increase in a measure of renalfunction. Preferred methods for measuring and/or estimating GFR aredescribed hereinafter.

As used herein, “reduced renal function” is an abrupt (within 14 days,preferably within 7 days, more preferably within 72 hours, and stillmore preferably within 48 hours) reduction in kidney function identifiedby an absolute increase in serum creatinine of greater than or equal to0.1 mg/dL (≥8.8 μmol/L), a percentage increase in serum creatinine ofgreater than or equal to 20% (1.2-fold from baseline), or a reduction inurine output (documented oliguria of less than 0.5 ml/kg per hour).

As used herein, “acute renal failure” or “ARF” is an abrupt (within 14days, preferably within 7 days, more preferably within 72 hours, andstill more preferably within 48 hours) reduction in kidney functionidentified by an absolute increase in serum creatinine of greater thanor equal to 0.3 mg/dl (≥26.4 μmol/L), a percentage increase in serumcreatinine of greater than or equal to 50% (1. 5-fold from baseline), ora reduction in urine output (documented oliguria of less than 0.5 ml/kgper hour for at least 6 hours). This term is synonymous with “acutekidney injury” or “AKI.”

As used herein, the term “Coagulation factor VII” refers to one or morepolypeptides present in a biological sample that are derived from theCoagulation factor VII precursor (human precursor: Swiss-Prot P08709(SEQ ID NO: 1))

        10         20         30         40         50         60MVSQALRLLC LLLGLQGCLA AGGVAKASGG ETRDMPWKPG PHRVFVTQEE AHGVLHRRRR        70         80         90        100        110        120ANAFLEELRP GSLERECKEE QCSFEEAREI FKDAERTKLF WISYSDGDQC ASSPCQNGGS       130        140        150        160        170        180CKDQLQSYIC FCLPAFEGRN CETHKDDQLI CVNENGGCEQ YCSDHTGTKR SCRCHEGYSL       190        200        210        220        230        240LADGVSCTPT VEYPCGKIPI LEKRNASKPQ GRIVGGKVCP KGECPWQVLL LVNGAQLCGG       250        260        270        280        290        300TLINTIWVVS AAHCFDKIKN WRNLIAVLGE HDLSEHDGDE QSRRVAQVII PSTYVPGTTN       310        320        330        340        350        360HDIALLRLHQ PVVLTDHVVP LCLPERTFSE RTLAFVRFSL VSGWGQLLDR GATALELMVL       370        380        390        400        410        420NVPRLMTQDC LQQSRKVGDS PNITEYMFCA GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG       430        440        450        460IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL MRSEPRPGVL LRAPFP

The following domains have been identified in Coagulation factor VII:

Residues Length Domain ID  1-20 20 Signal peptide 21-60 40 Propeptide 61-212 152 Coagulation factor VII, light chain 213-466 254 Coagulationfactor VII, heavy chain 22-43 22 Missing in Coagulation factor VIIisoform B

As used herein, the term “CA19-9” (also called carbohydrate antigen 19-9or sialylated Lewis (a) antigen) is a monosialoganglioside antigen foundin patients with gastrointestinal adenocarcinoma. It is reportedlyelevated in 21 to 42 percent of cases of gastric cancer, 20 to 40percent of colon cancer, and 71 to 93 percent of pancreatic cancer. Themain clinical use of CA19-9 is to see whether a pancreatic tumor issecreting it; if that is the case, then the levels should fall when thetumor is treated, and they may rise again if the disease recurs. In 5%of patients who lack the Lewis antigen, CA19-9 is not elevated inpancreatic cancer even with large tumors because they have a deficiencyof a fucosyltransferase enzyme that is needed to produce CA19-9 as wellas the Lewis antigen.

As used herein, the term “Insulin-like growth factor-binding protein 7”refers to one or more polypeptides present in a biological sample thatare derived from the Insulin-like growth factor-binding protein 7precursor (human precursor: Swiss-Prot Q16270 (SEQ ID NO: 2))

        10         20         30         40         50         60MERPSLRALL LGAAGLLLLL LPLSSSSSSD TCGPCEPASC PPLPPLGCLL GETRDACGCC        70         80         90        100        110        120PMCARGEGEP CGGGGAGRGY CAPGMECVKS RKRRKGKAGA AAGGPGVSGV CVCKSRYPVC       130        140        150        160        170        180GSDGTTYPSG CQLRAASQRA ESRGEKAITQ VSKGTCEQGP SIVTPPKDIW NVTGAQVYLS       190        200        210        220        230        240CEVIGIPTPV LIWNKVKRGH YGVQRTELLP GDRDNLAIQT RGGPEKHEVT GWVLVSPLSK       250        260        270        280EDAGEYECHA SNSQGQASAS AKITVVDALH EIPVKKGEGA EL

The following domains have been identified in Insulin-like growthfactor-binding protein 7:

Residues Length Domain ID 1-26 26 Signal peptide 27-282 256 Insulin-likegrowth factor-binding protein 7

As used herein, the term “C—X—C motif chemokine 6” refers to one or morepolypeptides present in a biological sample that are derived from theC—X—C motif chemokine 6 precursor (human precursor: Swiss-Prot P80162(SEQ ID NO: 3))

        10         20         30         40         50         60MSLPSSRAAR VPGPSGSLCA LLALLLLLTP PGPLASAGPV SAVLTELRCT CLRVTLRVNP        70         80         90        100        110KTIGKLQVFP AGPQCSKVEV VASLKNGKQV CLDPEAPFLK KVIQKILDSG NKKN

The following domains have been identified in C—X—C motif chemokine 6:

Residues Length Domain ID 1-37 37 Signal peptide 38-114 77 C-X-C motifchemokine 6 40-114 75 C-X-C motif chemokine 6 (N-processed variant 1)43-114 72 C-X-C motif chemokine 6 (N-processed variant 2) 46-114 69C-X-C motif chemokine 6 (N-processed variant 3)

As used herein, the term “C—C motif chemokine 13” refers to one or morepolypeptides present in a biological sample that are derived from theC—C motif chemokine 13 precursor (human precursor: Swiss-Prot Q99616(SEQ ID NO: 4))

        10         20         30         40         50         60MKVSAVLLCL LLMTAAFNPQ GLAQPDALNV PSTCCFTFSS KKISLQRLKS YVITTSRCPQ        70         80         90KAVIFRTKLG KEICADPKEK WVQNYMKHLG RKAHTLKT

The following domains have been identified in C—C motif chemokine 13:

Residues Length Domain ID  1-16 16 Signal peptide 17-98 82 C-C motifchemokine 13, long chain 22-98 82 C-C motif chemokine 13, medium chain24-98 82 C-C motif chemokine 13, short chain

As used herein, the term “relating a signal to the presence or amount”of an analyte reflects the following understanding. Assay signals aretypically related to the presence or amount of an analyte through theuse of a standard curve calculated using known concentrations of theanalyte of interest. As the term is used herein, an assay is “configuredto detect” an analyte if an assay can generate a detectable signalindicative of the presence or amount of a physiologically relevantconcentration of the analyte. Because an antibody epitope is on theorder of 8 amino acids, an immunoassay configured to detect a marker ofinterest will also detect polypeptides related to the marker sequence,so long as those polypeptides contain the epitope(s) necessary to bindto the antibody or antibodies used in the assay. The term “relatedmarker” as used herein with regard to a biomarker such as one of thekidney injury markers described herein refers to one or more fragments,variants, etc., of a particular marker or its biosynthetic parent thatmay be detected as a surrogate for the marker itself or as independentbiomarkers. The term also refers to one or more polypeptides present ina biological sample that are derived from the biomarker precursorcomplexed to additional species, such as binding proteins, receptors,heparin, lipids, sugars, etc.

In this regard, the skilled artisan will understand that the signalsobtained from an immunoassay are a direct result of complexes formedbetween one or more antibodies and the target biomolecule (i.e., theanalyte) and polypeptides containing the necessary epitope(s) to whichthe antibodies bind. While such assays may detect the full lengthbiomarker and the assay result be expressed as a concentration of abiomarker of interest, the signal from the assay is actually a result ofall such “immunoreactive” polypeptides present in the sample. Expressionof biomarkers may also be determined by means other than immunoassays,including protein measurements (such as dot blots, western blots,chromatographic methods, mass spectrometry, etc.) and nucleic acidmeasurements (mRNA quatitation). This list is not meant to be limiting.

The term “positive going” marker as that term is used herein refer to amarker that is determined to be elevated in subjects suffering from adisease or condition, relative to subjects not suffering from thatdisease or condition. The term “negative going” marker as that term isused herein refer to a marker that is determined to be reduced insubjects suffering from a disease or condition, relative to subjects notsuffering from that disease or condition.

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects arehumans, and most preferably “patients,” which as used herein refers toliving humans that are receiving medical care for a disease orcondition. This includes persons with no defined illness who are beinginvestigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may beobtained from a subject, or may be obtained from biological materialsintended to be provided to the subject. For example, a sample may beobtained from a kidney being evaluated for possible transplantation intoa subject, and an analyte measurement used to evaluate the kidney forpreexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodilyfluid obtained for the purpose of diagnosis, prognosis, classificationor evaluation of a subject of interest, such as a patient or transplantdonor. In certain embodiments, such a sample may be obtained for thepurpose of determining the outcome of an ongoing condition or the effectof a treatment regimen on a condition. Preferred body fluid samplesinclude blood, serum, plasma, cerebrospinal fluid, urine, saliva,sputum, and pleural effusions. In addition, one of skill in the artwould realize that certain body fluid samples would be more readilyanalyzed following a fractionation or purification procedure, forexample, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine the probability (“alikelihood”) of whether or not a patient is suffering from a givendisease or condition. In the case of the present invention, “diagnosis”includes using the results of an assay, most preferably an immunoassay,for a kidney injury marker of the present invention, optionally togetherwith other clinical characteristics, to arrive at a diagnosis (that is,the occurrence or nonoccurrence) of an acute renal injury or ARF for thesubject from which a sample was obtained and assayed. That such adiagnosis is “determined” is not meant to imply that the diagnosis is100% accurate. Many biomarkers are indicative of multiple conditions.The skilled clinician does not use biomarker results in an informationalvacuum, but rather test results are used together with other clinicalindicia to arrive at a diagnosis. Thus, a measured biomarker level onone side of a predetermined diagnostic threshold indicates a greaterlikelihood of the occurrence of disease in the subject relative to ameasured level on the other side of the predetermined diagnosticthreshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) thata given course or outcome will occur. A level or a change in level of aprognostic indicator, which in turn is associated with an increasedprobability of morbidity (e.g., worsening renal function, future ARF, ordeath) is referred to as being “indicative of an increased likelihood”of an adverse outcome in a patient.

Marker Assays

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include those developed and/or usedas solid phases in solid phase binding assays. Examples of suitablesolid phases include membrane filters, cellulose-based papers, beads(including polymeric, latex and paramagnetic particles), glass, siliconwafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels,SPOCC gels, and multiple-well plates. An assay strip could be preparedby coating the antibody or a plurality of antibodies in an array onsolid support. This strip could then be dipped into the test sample andthen processed quickly through washes and detection steps to generate ameasurable signal, such as a colored spot. Antibodies or otherpolypeptides may be bound to specific zones of assay devices either byconjugating directly to an assay device surface, or by indirect binding.In an example of the later case, antibodies or other polypeptides may beimmobilized on particles or other solid supports, and that solid supportimmobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react non-specifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysisof the described kidney injury markers. The kit comprises reagents forthe analysis of at least one test sample which comprise at least oneantibody that a kidney injury marker. The kit can also include devicesand instructions for performing one or more of the diagnostic and/orprognostic correlations described herein. Preferred kits will comprisean antibody pair for performing a sandwich assay, or a labeled speciesfor performing a competitive assay, for the analyte. Preferably, anantibody pair comprises a first antibody conjugated to a solid phase anda second antibody conjugated to a detectable label, wherein each of thefirst and second antibodies that bind a kidney injury marker. Mostpreferably each of the antibodies are monoclonal antibodies. Theinstructions for use of the kit and performing the correlations can bein the form of labeling, which refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabeling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Antibodies used in the immunoassays described herein preferablyspecifically bind to a kidney injury marker of the present invention.The term “specifically binds” is not intended to indicate that anantibody binds exclusively to its intended target since, as noted above,an antibody binds to any polypeptide displaying the epitope(s) to whichthe antibody binds. Rather, an antibody “specifically binds” if itsaffinity for its intended target is about 5-fold greater when comparedto its affinity for a non-target molecule which does not display theappropriate epitope(s). Preferably the affinity of the antibody will beat least about 5 fold, preferably 10 fold, more preferably 25-fold, evenmore preferably 50-fold, and most preferably 100-fold or more, greaterfor a target molecule than its affinity for a non-target molecule. Inpreferred embodiments, Preferred antibodies bind with affinities of atleast about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ m⁻¹ to about 10¹²M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, K_(on) is the association rate constant andK_(d) is the equilibrium constant). Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor atequilibrium; c=free ligand concentration at equilibrium; K=equilibriumassociation constant; and n=number of ligand binding sites per receptormolecule. By graphical analysis, r/c is plotted on the Y-axis versus ron the X-axis, thus producing a Scatchard plot. Antibody affinitymeasurement by Scatchard analysis is well known in the art. See, e.g.,van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold,Comput. Methods Programs Biomed. 27: 65-8, 1988.

The term “epitope” refers to an antigenic determinant capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

Numerous publications discuss the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedanalyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. Abasic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the polypeptide. This physical association is provided by the phageparticle, which displays a polypeptide as part of a capsid enclosing thephage genome which encodes the polypeptide. The establishment of aphysical association between polypeptides and their genetic materialallows simultaneous mass screening of very large numbers of phagebearing different polypeptides. Phage displaying a polypeptide withaffinity to a target bind to the target and these phage are enriched byaffinity screening to the target. The identity of polypeptides displayedfrom these phage can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in itsentirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

While the present application describes antibody-based binding assays indetail, alternatives to antibodies as binding species in assays are wellknown in the art. These include receptors for a particular target,aptamers, etc. Aptamers are oligonucleic acid or peptide molecules thatbind to a specific target molecule. Aptamers are usually created byselecting them from a large random sequence pool, but natural aptamersalso exist. High-affinity aptamers containing modified nucleotidesconferring improved characteristics on the ligand, such as improved invivo stability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions, and may include amino acid side chainfunctionalities.

Assay Correlations

The term “correlating” as used herein in reference to the use ofbiomarkers refers to comparing the presence or amount of thebiomarker(s) in a patient to its presence or amount in persons known tosuffer from, or known to be at risk of, a given condition; or in personsknown to be free of a given condition. Often, this takes the form ofcomparing an assay result in the form of a biomarker concentration to apredetermined threshold selected to be indicative of the occurrence ornonoccurrence of a disease or the likelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things,consideration of the probability of disease, distribution of true andfalse diagnoses at different test thresholds, and estimates of theconsequences of treatment (or a failure to treat) based on thediagnosis. For example, when considering administering a specifictherapy which is highly efficacious and has a low level of risk, fewtests are needed because clinicians can accept substantial diagnosticuncertainty. On the other hand, in situations where treatment optionsare less effective and more risky, clinicians often need a higher degreeof diagnostic certainty. Thus, cost/benefit analysis is involved inselecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example,one recommended diagnostic threshold for the diagnosis of acutemyocardial infarction using cardiac troponin is the 97.5th percentile ofthe concentration seen in a normal population. Another method may be tolook at serial samples from the same patient, where a prior “baseline”result is used to monitor for temporal changes in a biomarker level.

Population studies may also be used to select a decision threshold.Reciever Operating Characteristic (“ROC”) arose from the field of signaldectection therory developed during World War II for the analysis ofradar images, and ROC analysis is often used to select a threshold ableto best distinguish a “diseased” subpopulation from a “nondiseased”subpopulation. A false positive in this case occurs when the persontests positive, but actually does not have the disease. A falsenegative, on the other hand, occurs when the person tests negative,suggesting they are healthy, when they actually do have the disease. Todraw a ROC curve, the true positive rate (TPR) and false positive rate(FPR) are determined as the decision threshold is varied continuously.Since TPR is equivalent with sensitivity and FPR is equal to1-specificity, the ROC graph is sometimes called the sensitivity vs(1-specificity) plot. A perfect test will have an area under the ROCcurve of 1.0; a random test will have an area of 0.5. A threshold isselected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having onecharacteristic (the presence of a disease or condition or the occurrenceof some outcome) and “nondiseased” is meant to refer to a populationlacking the characteristic. While a single decision threshold is thesimplest application of such a method, multiple decision thresholds maybe used. For example, below a first threshold, the absence of diseasemay be assigned with relatively high confidence, and above a secondthreshold the presence of disease may also be assigned with relativelyhigh confidence. Between the two thresholds may be consideredindeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural network methods. These methods canproduce probability values representing the degree to which a subjectbelongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer etal., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given biomarker. These measures include sensitivityand specificity, predictive values, likelihood ratios, diagnostic oddsratios, and ROC curve areas. The area under the curve (“AUC”) of a ROCplot is equal to the probability that a classifier will rank a randomlychosen positive instance higher than a randomly chosen negative one. Thearea under the ROC curve may be thought of as equivalent to theMann-Whitney U test, which tests for the median difference betweenscores obtained in the two groups considered if the groups are ofcontinuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of thefollowing results on these various measures: a specificity of greaterthan 0.5, preferably at least 0.6, more preferably at least 0.7, stillmore preferably at least 0.8, even more preferably at least 0.9 and mostpreferably at least 0.95, with a corresponding sensitivity greater than0.2, preferably greater than 0.3, more preferably greater than 0.4,still more preferably at least 0.5, even more preferably 0.6, yet morepreferably greater than 0.7, still more preferably greater than 0.8,more preferably greater than 0.9, and most preferably greater than 0.95;a sensitivity of greater than 0.5, preferably at least 0.6, morepreferably at least 0.7, still more preferably at least 0.8, even morepreferably at least 0.9 and most preferably at least 0.95, with acorresponding specificity greater than 0.2, preferably greater than 0.3,more preferably greater than 0.4, still more preferably at least 0.5,even more preferably 0.6, yet more preferably greater than 0.7, stillmore preferably greater than 0.8, more preferably greater than 0.9, andmost preferably greater than 0.95; at least 75% sensitivity, combinedwith at least 75% specificity; a ROC curve area of greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95; an odds ratio different from 1, preferably at least about 2or more or about 0.5 or less, more preferably at least about 3 or moreor about 0.33 or less, still more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less; a positive likelihood ratio (calculated assensitivity/(1-specificity)) of greater than 1, at least 2, morepreferably at least 3, still more preferably at least 5, and mostpreferably at least 10; and or a negative likelihood ratio (calculatedas (1-sensitivity)/specificity) of less than 1, less than or equal to0.5, more preferably less than or equal to 0.3, and most preferably lessthan or equal to 0.1

Additional clinical indicia may be combined with the kidney injurymarker assay result(s) of the present invention. These include otherbiomarkers related to renal status. Examples include the following,which recite the common biomarker name, followed by the Swiss-Prot entrynumber for that biomarker or its parent: Actin (P68133); Adenosinedeaminase binding protein (DPP4, P27487); Alpha-1-acid glycoprotein 1(P02763); Alpha-1-microglobulin (P02760); Albumin (P02768);Angiotensinogenase (Renin, P00797); Annexin A2 (P07355);Beta-glucuronidase (P08236); B-2-microglobulin (P61679);Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide(proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta(S100-beta, P04271); Carbonic anhydrase (Q16790); Casein Kinase 2(P68400); Ceruloplasmin (P00450); Clusterin (P10909); Complement C3(P01024); Cysteine-rich protein (CYR61, O00622); Cytochrome C(P99999);Epidermal growth factor (EGF, P01133); Endothelin-1 (P05305); ExosomalFetuin-A (P02765); Fatty acid-binding protein, heart (FABP3, P05413);Fatty acid-binding protein, liver (P07148); Ferritin (light chain,P02793; heavy chain P02794); Fructose-1,6-biphosphatase (P09467);GRO-alpha (CXCL1, (P09341); Growth Hormone (P01241); Hepatocyte growthfactor (P14210); Insulin-like growth factor I (P01343); ImmunoglobulinG; Immunoglobulin Light Chains (Kappa and Lambda); Interferon gamma(P01308); Lysozyme (P61626); Interleukin-1alpha (P01583); Interleukin-2(P60568); Interleukin-4 (P60568); Interleukin-9 (P15248);Interleukin-12p40 (P29460); Interleukin-13 (P35225); Interleukin-16(Q14005); L1 cell adhesion molecule (P32004); Lactate dehydrogenase(P00338); Leucine Aminopeptidase (P28838); Meprin A-alpha subunit(Q16819); Meprin A-beta subunit (Q16820); Midkine (P21741); MIP2-alpha(CXCL2, P19875); MMP-2 (P08253); MMP-9 (P14780); Netrin-1 (O95631);Neutral endopeptidase (P08473); Osteopontin (P10451); Renal papillaryantigen 1 (RPA1); Renal papillary antigen 2 (RPA2); Retinol bindingprotein (P09455); Ribonuclease; S100 calcium-binding protein A6(P06703); Serum Amyloid P Component (P02743); Sodium/Hydrogen exchangerisoform (NHE3, P48764); Spermidine/spermine N1-acetyltransferase(P21673); TGF-Beta1 (P01137); Transferrin (P02787); Trefoil factor 3(TFF3, Q07654); Toll-Like protein 4 (O00206); Total protein;Tubulointerstitial nephritis antigen (Q9UJW2); Uromodulin (Tamm-Horsfallprotein, P07911).

For purposes of risk stratification, Adiponectin (Q15848); Alkalinephosphatase (P05186); Aminopeptidase N(P15144); CalbindinD28k (P05937);Cystatin C(P01034); 8 subunit of FIFO ATPase (P03928);Gamma-glutamyltransferase (P19440); GSTa(alpha-glutathione-S-transferase, P08263); GSTpi(Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1 (P08833);IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itml,P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18(Q14116); IP-10 (10 kDa interferon-gamma-induced protein, P02778); IRPR(IFRD1, O00458); Isovaleryl-CoA dehydrogenase (IVD, P26440);I-TAC/CXCL11 (O14625); Keratin 19 (P08727); Kim-1 (Hepatitis A viruscellular receptor 1, O43656); L-arginine:glycine amidinotransferase(P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500);MIG (Gamma-interferon-induced monokine Q07325); MIP-1a (P10147); MIP-3a(P78556); MIP-1beta (P13236); MIP-1d (Q16663); NAG(N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter(OCT2, O15244); Osteoprotegerin (O14788); P8 protein (O60356);Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP(1-98)(P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta(P50395); Renal kallikrein (Q86U61); RT1.B-1 (alpha) chain of theintegral membrane protein (Q5Y7A8); Soluble tumor necrosis factorreceptor superfamily member 1A (sTNFR-I, P19438); Soluble tumor necrosisfactor receptor superfamily member 1B (sTNFR-II, P20333); Tissueinhibitor of metalloproteinases 3 (TIMP-3, P35625); uPAR (Q03405) may becombined with the kidney injury marker assay result(s) of the presentinvention.

Other clinical indicia which may be combined with the kidney injurymarker assay result(s) of the present invention includes demographicinformation (e.g., weight, sex, age, race), medical history (e.g.,family history, type of surgery, pre-existing disease such as aneurism,congestive heart failure, preeclampsia, eclampsia, diabetes mellitus,hypertension, coronary artery disease, proteinuria, renal insufficiency,or sepsis, type of toxin exposure such as NSAIDs, cyclosporines,tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin,myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrastagents, or streptozotocin), clinical variables (e.g., blood pressure,temperature, respiration rate), risk scores (APACHE score, PREDICTscore, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urinetotal protein measurement, a glomerular filtration rate, an estimatedglomerular filtration rate, a urine production rate, a serum or plasmacreatinine concentration, a renal papillary antigen 1 (RPA1)measurement; a renal papillary antigen 2 (RPA2) measurement; a urinecreatinine concentration, a fractional excretion of sodium, a urinesodium concentration, a urine creatinine to serum or plasma creatinineratio, a urine specific gravity, a urine osmolality, a urine ureanitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio,and/or a renal failure index calculated as urine sodium/(urinecreatinine/plasma creatinine). Other measures of renal function whichmay be combined with the kidney injury marker assay result(s) aredescribed hereinafter and in Harrison's Principles of Internal Medicine,17^(th) Ed., McGraw Hill, New York, pages 1741-1830, and Current MedicalDiagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, New York, pages785-815, each of which are hereby incorporated by reference in theirentirety.

Combining assay results/clinical indicia in this manner can comprise theuse of multivariate logistical regression, loglinear modeling, neuralnetwork analysis, n-of-m analysis, decision tree analysis, etc. Thislist is not meant to be limiting.

Diagnosis of Acute Renal Failure

As noted above, the terms “acute renal (or kidney) injury” and “acuterenal (or kidney) failure” as used herein are defined in part in termsof changes in serum creatinine from a baseline value. Most definitionsof ARF have common elements, including the use of serum creatinine and,often, urine output. Patients may present with renal dysfunction withoutan available baseline measure of renal function for use in thiscomparison. In such an event, one may estimate a baseline serumcreatinine value by assuming the patient initially had a normal GFR.Glomerular filtration rate (GFR) is the volume of fluid filtered fromthe renal (kidney) glomerular capillaries into the Bowman's capsule perunit time. Glomerular filtration rate (GFR) can be calculated bymeasuring any chemical that has a steady level in the blood, and isfreely filtered but neither reabsorbed nor secreted by the kidneys. GFRis typically expressed in units of ml/min:

${G\; F\; R} = \frac{{Urine}\mspace{14mu}{Concentration} \times {Urine}\mspace{14mu}{Flow}}{{Plasma}\mspace{14mu}{Concentration}}$

By normalizing the GFR to the body surface area, a GFR of approximately75-100 ml/min per 1.73 m² can be assumed. The rate therefore measured isthe quantity of the substance in the urine that originated from acalculable volume of blood.

There are several different techniques used to calculate or estimate theglomerular filtration rate (GFR or eGFR). In clinical practice, however,creatinine clearance is used to measure GFR. Creatinine is producednaturally by the body (creatinine is a metabolite of creatine, which isfound in muscle). It is freely filtered by the glomerulus, but alsoactively secreted by the renal tubules in very small amounts such thatcreatinine clearance overestimates actual GFR by 10-20%. This margin oferror is acceptable considering the ease with which creatinine clearanceis measured.

Creatinine clearance (CCr) can be calculated if values for creatinine'surine concentration (U_(Cr)), urine flow rate (V), and creatinine'splasma concentration (P_(Cr)) are known. Since the product of urineconcentration and urine flow rate yields creatinine's excretion rate,creatinine clearance is also said to be its excretion rate (U_(Cr)×V)divided by its plasma concentration. This is commonly representedmathematically as:

$C_{Cr} = \frac{U_{Cr} - V}{P_{Cr}}$

Commonly a 24 hour urine collection is undertaken, from empty-bladderone morning to the contents of the bladder the following morning, with acomparative blood test then taken:

$C_{Cr} = \frac{U_{Cr} \times 24\text{-}{hour}\mspace{14mu}{volume}}{P_{Cr} \times 24 \times 60\mspace{14mu}{mins}}$

To allow comparison of results between people of different sizes, theCCr is often corrected for the body surface area (BSA) and expressedcompared to the average sized man as ml/min/1.73 m2. While most adultshave a BSA that approaches 1.7 (1.6-1.9), extremely obese or slimpatients should have their CCr corrected for their actual BSA:

$C_{{Cr} - {corrected}} = \frac{C_{Cr} \times 1.73`}{B\; S\; A}$

The accuracy of a creatinine clearance measurement (even when collectionis complete) is limited because as glomerular filtration rate (GFR)falls creatinine secretion is increased, and thus the rise in serumcreatinine is less. Thus, creatinine excretion is much greater than thefiltered load, resulting in a potentially large overestimation of theGFR (as much as a twofold difference). However, for clinical purposes itis important to determine whether renal function is stable or gettingworse or better. This is often determined by monitoring serum creatininealone. Like creatinine clearance, the serum creatinine will not be anaccurate reflection of GFR in the non-steady-state condition of ARF.Nonetheless, the degree to which serum creatinine changes from baselinewill reflect the change in GFR. Serum creatinine is readily and easilymeasured and it is specific for renal function.

For purposes of determining urine output on a Urine output on a mL/kg/hrbasis, hourly urine collection and measurement is adequate. In the casewhere, for example, only a cumulative 24-h output was available and nopatient weights are provided, minor modifications of the RIFLE urineoutput criteria have been described. For example, Bagshaw et al.,Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an averagepatient weight of 70 kg, and patients are assigned a RIFLEclassification based on the following: <35 mL/h (Risk), <21 mL/h(Injury) or <4 mL/h (Failure).

Selecting a Treatment Regimen

Once a diagnosis is obtained, the clinician can readily select atreatment regimen that is compatible with the diagnosis, such asinitiating renal replacement therapy, withdrawing delivery of compoundsthat are known to be damaging to the kidney, kidney transplantation,delaying or avoiding procedures that are known to be damaging to thekidney, modifying diuretic administration, initiating goal directedtherapy, etc. The skilled artisan is aware of appropriate treatments fornumerous diseases discussed in relation to the methods of diagnosisdescribed herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17thEd. Merck Research Laboratories, Whitehouse Station, N.J., 1999. Inaddition, since the methods and compositions described herein provideprognostic information, the markers of the present invention may be usedto monitor a course of treatment. For example, improved or worsenedprognostic state may indicate that a particular treatment is or is notefficacious.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

Example 1 Contrast-Induced Nephropathy Sample Collection

The objective of this sample collection study is to collect samples ofplasma and urine and clinical data from patients before and afterreceiving intravascular contrast media. Approximately 250 adultsundergoing radiographic/angiographic procedures involving intravascularadministration of iodinated contrast media are enrolled. To be enrolledin the study, each patient must meet all of the following inclusioncriteria and none of the following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older;

undergoing a radiographic/angiographic procedure (such as a CT scan orcoronary intervention) involving the intravascular administration ofcontrast media;

expected to be hospitalized for at least 48 hours after contrastadministration.

able and willing to provide written informed consent for studyparticipation and to comply with all study procedures.

Exclusion Criteria

renal transplant recipients;

acutely worsening renal function prior to the contrast procedure;

already receiving dialysis (either acute or chronic) or in imminent needof dialysis at enrollment;

expected to undergo a major surgical procedure (such as involvingcardiopulmonary bypass) or an additional imaging procedure with contrastmedia with significant risk for further renal insult within the 48 hrsfollowing contrast administration;

participation in an interventional clinical study with an experimentaltherapy within the previous 30 days;

known infection with human immunodeficiency virus (HIV) or a hepatitisvirus.

Immediately prior to the first contrast administration (and after anypre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL)and a urine sample (10 mL) are collected from each patient. Blood andurine samples are then collected at 4 (±0.5), 8 (±1), 24 (±2) 48 (±2),and 72 (±2) hrs following the last administration of contrast mediaduring the index contrast procedure. Blood is collected via directvenipuncture or via other available venous access, such as an existingfemoral sheath, central venous line, peripheral intravenous line orhep-lock. These study blood samples are processed to plasma at theclinical site, frozen and shipped to Astute Medical, Inc., San Diego,Calif. The study urine samples are frozen and shipped to Astute Medical,Inc.

Serum creatinine is assessed at the site immediately prior to the firstcontrast administration (after any pre-procedure hydration) and at 4(±0.5), 8 (±1), 24 (±2) and 48 (±2)), and 72 (±2) hours following thelast administration of contrast (ideally at the same time as the studysamples are obtained). In addition, each patient's status is evaluatedthrough day 30 with regard to additional serum and urine creatininemeasurements, a need for dialysis, hospitalization status, and adverseclinical outcomes (including mortality).

Prior to contrast administration, each patient is assigned a risk basedon the following assessment: systolic blood pressure <80 mm Hg=5 points;intra-arterial balloon pump=5 points; congestive heart failure (ClassIII-IV or history of pulmonary edema)=5 points; age >75 yrs=4 points;hematocrit level <39% for men, <35% for women=3 points; diabetes=3points; contrast media volume=1 point for each 100 mL; serum creatininelevel >1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m²=2 points,20-40 mL/min/1.73 m²=4 points, <20 mL/min/1.73 m²=6 points. The risksassigned are as follows: risk for CIN and dialysis: 5 or less totalpoints=risk of CIN-7.5%, risk of dialysis-0.04%; 6-10 total points=riskof CIN-14%, risk of dialysis-0.12%; 11-16 total points=risk ofCIN-26.1%, risk of dialysis-1.09%; >16 total points=risk of CIN-57.3%,risk of dialysis-12.8%.

Example 2 Cardiac Surgery Sample Collection

The objective of this sample collection study is to collect samples ofplasma and urine and clinical data from patients before and afterundergoing cardiovascular surgery, a procedure known to be potentiallydamaging to kidney function. Approximately 900 adults undergoing suchsurgery are enrolled. To be enrolled in the study, each patient mustmeet all of the following inclusion criteria and none of the followingexclusion criteria:

Inclusion Criteria

males and females 18 years of age or older;

undergoing cardiovascular surgery;

Toronto/Ottawa Predictive Risk Index for Renal Replacement risk score ofat least 2 (Wijeysundera et al., JAMA 297: 1801-9, 2007); and

able and willing to provide written informed consent for studyparticipation and to comply with all study procedures.

Exclusion Criteria

known pregnancy;

previous renal transplantation;

acutely worsening renal function prior to enrollment (e.g., any categoryof RIFLE criteria);

already receiving dialysis (either acute or chronic) or in imminent needof dialysis at enrollment;

currently enrolled in another clinical study or expected to be enrolledin another clinical study within 7 days of cardiac surgery that involvesdrug infusion or a therapeutic intervention for AKI;

known infection with human immunodeficiency virus (HIV) or a hepatitisvirus.

Within 3 hours prior to the first incision (and after any pre-procedurehydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3mL), and a urine sample (35 mL) are collected from each patient. Bloodand urine samples are then collected at 3 (±0.5), 6 (±0.5), 12 (±1), 24(±2) and 48 (±2) hrs following the procedure and then daily on days 3through 7 if the subject remains in the hospital. Blood is collected viadirect venipuncture or via other available venous access, such as anexisting femoral sheath, central venous line, peripheral intravenousline or hep-lock. These study blood samples are frozen and shipped toAstute Medical, Inc., San Diego, Calif. The study urine samples arefrozen and shipped to Astute Medical, Inc.

Example 3 Acutely Ill Subject Sample Collection

The objective of this study is to collect samples from acutely illpatients. Approximately 900 adults expected to be in the ICU for atleast 48 hours will be enrolled. To be enrolled in the study, eachpatient must meet all of the following inclusion criteria and none ofthe following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older;

Study population 1: approximately 300 patients that have at least oneof:

shock (SBP <90 mmHg and/or need for vasopressor support to maintainMAP >60 mmHg and/or documented drop in SBP of at least 40 mmHg); andsepsis;

Study population 2: approximately 300 patients that have at least oneof:

IV antibiotics ordered in computerized physician order entry (CPOE)within 24 hours of enrollment;

contrast media exposure within 24 hours of enrollment;

increased Intra-Abdominal Pressure with acute decompensated heartfailure; and

severe trauma as the primary reason for ICU admission and likely to behospitalized in the ICU for 48 hours after enrollment;

Study population 3: approximately 300 patients expected to behospitalized through acute care setting (ICU or ED) with a known riskfactor for acute renal injury (e.g. sepsis, hypotension/shock(Shock=systolic BP<90 mmHg and/or the need for vasopressor support tomaintain a MAP >60 mmHg and/or a documented drop in SBP >40 mmHg), majortrauma, hemorrhage, or major surgery); and/or expected to behospitalized to the ICU for at least 24 hours after enrollment.Exclusion Criteriaknown pregnancy;institutionalized individuals;previous renal transplantation;known acutely worsening renal function prior to enrollment (e.g., anycategory of RIFLE criteria);received dialysis (either acute or chronic) within 5 days prior toenrollment or in imminent need of dialysis at the time of enrollment;known infection with human immunodeficiency virus (HIV) or a hepatitisvirus;meets only the SBP <90 mmHg inclusion criterion set forth above, anddoes not have shock in the attending physician's or principalinvestigator's opinion.

After providing informed consent, an EDTA anti-coagulated blood sample(10 mL) and a urine sample (25-30 mL) are collected from each patient.Blood and urine samples are then collected at 4 (±0.5) and 8 (±1) hoursafter contrast administration (if applicable); at 12 (±1), 24 (±2), and48 (±2) hours after enrollment, and thereafter daily up to day 7 to day14 while the subject is hospitalized. Blood is collected via directvenipuncture or via other available venous access, such as an existingfemoral sheath, central venous line, peripheral intravenous line orhep-lock. These study blood samples are processed to plasma at theclinical site, frozen and shipped to Astute Medical, Inc., San Diego,Calif. The study urine samples are frozen and shipped to Astute Medical,Inc.

Example 4 Immunoassay Format

Analytes are measured using standard sandwich enzyme immunoassaytechniques. A first antibody which binds the analyte is immobilized inwells of a 96 well polystyrene microplate. Analyte standards and testsamples are pipetted into the appropriate wells and any analyte presentis bound by the immobilized antibody. After washing away any unboundsubstances, a horseradish peroxidase-conjugated second antibody whichbinds the analyte is added to the wells, thereby forming sandwichcomplexes with the analyte (if present) and the first antibody.Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution comprising tetramethylbenzidine and hydrogen peroxideis added to the wells. Color develops in proportion to the amount ofanalyte present in the sample. The color development is stopped and theintensity of the color is measured at 540 nm or 570 nm. An analyteconcentration is assigned to the test sample by comparison to a standardcurve determined from the analyte standards. Concentrations reportedbelow are as follows: Coagulation factor VII-ng/mL, CA19-9-U/mL,Insulin-like growth factor-binding protein 7 ng/mL, C—X—C motifchemokine 6-pg/mL, and C—C motif chemokine 13-pg/mL.

Example 5 Apparently Healthy Donor and Chronic Disease Patient Samples

Human urine samples from donors with no known chronic or acute disease(“Apparently Healthy Donors”) were purchased from two vendors (GoldenWest Biologicals, Inc., 27625 Commerce Center Dr., Temecula, Calif.92590 and Virginia Medical Research, Inc., 915 First Colonial Rd.,Virginia Beach, Va. 23454). The urine samples were shipped and storedfrozen at less than −20° C. The vendors supplied demographic informationfor the individual donors including gender, race (Black/White), smokingstatus and age.

Human urine samples from donors with various chronic diseases (“ChronicDisease Patients”) including congestive heart failure, coronary arterydisease, chronic kidney disease, chronic obstructive pulmonary disease,diabetes mellitus and hypertension were purchased from Virginia MedicalResearch, Inc., 915 First Colonial Rd., Virginia Beach, Va. 23454. Theurine samples were shipped and stored frozen at less than −20 degreescentigrade. The vendor provided a case report form for each individualdonor with age, gender, race (Black/White), smoking status and alcoholuse, height, weight, chronic disease(s) diagnosis, current medicationsand previous surgeries.

Example 6 Use of Kidney Injury Markers for Evaluating Renal Status inPatients

Patients from the intensive care unit (ICU) were enrolled in thefollowing study. Each patient was classified by kidney status asnon-injury (O), risk of injury (R), injury (I), and failure (F)according to the maximum stage reached within 7 days of enrollment asdetermined by the RIFLE criteria. EDTA anti-coagulated blood samples (10mL) and a urine samples (25-30 mL) were collected from each patient atenrollment, 4 (±0.5) and 8 (±1) hours after contrast administration (ifapplicable); at 12 (±1), 24 (±2), and 48 (±2) hours after enrollment,and thereafter daily up to day 7 to day 14 while the subject ishospitalized. Markers were each measured by standard immunoassay methodsusing commercially available assay reagents in the urine samples and theplasma component of the blood samples collected.

Two cohorts were defined to represent a “diseased” and a “normal”population. While these terms are used for convenience, “diseased” and“normal” simply represent two cohorts for comparison (say RIFLE 0 vsRIFLE R, I and F; RIFLE 0 vs RIFLE R; RIFLE 0 and R vs RIFLE I and F;etc.). The time “prior max stage” represents the time at which a sampleis collected, relative to the time a particular patient reaches thelowest disease stage as defined for that cohort, binned into threegroups which are +/−12 hours. For example, “24 hr prior” which uses 0 vsR, I, F as the two cohorts would mean 24 hr (+/−12 hours) prior toreaching stage R (or I if no sample at R, or F if no sample at R or I).

A receiver operating characteristic (ROC) curve was generated for eachbiomarker measured and the area under each ROC curve (AUC) isdetermined. Patients in Cohort 2 were also separated according to thereason for adjudication to cohort 2 as being based on serum creatininemeasurements (sCr), being based on urine output (UO), or being based oneither serum creatinine measurements or urine output. Using the sameexample discussed above (0 vs R, I, F), for those patients adjudicatedto stage R, I, or F on the basis of serum creatinine measurements alone,the stage 0 cohort may include patients adjudicated to stage R, I, or Fon the basis of urine output; for those patients adjudicated to stage R,I, or F on the basis of urine output alone, the stage 0 cohort mayinclude patients adjudicated to stage R, I, or F on the basis of serumcreatinine measurements; and for those patients adjudicated to stage R,I, or F on the basis of serum creatinine measurements or urine output,the stage 0 cohort contains only patients in stage 0 for both serumcreatinine measurements and urine output. Also, in the data for patientsadjudicated on the basis of serum creatinine measurements or urineoutput, the adjudication method which yielded the most severe RIFLEstage is used.

The ability to distinguish cohort 1 from Cohort 2 was determined usingROC analysis. SE is the standard error of the AUC, n is the number ofsample or individual patients (“pts,” as indicated). Standard errors arecalculated as described in Hanley, J. A., and McNeil, B. J., The meaningand use of the area under a receiver operating characteristic (ROC)curve. Radiology (1982) 143: 29-36; p values are calculated with atwo-tailed Z-test. An AUC <0.5 is indicative of a negative going markerfor the comparison, and an AUC >0.5 is indicative of a positive goingmarker for the comparison.

Various threshold (or “cutoff”) concentrations were selected, and theassociated sensitivity and specificity for distinguishing cohort 1 fromcohort 2 are determined. OR is the odds ratio calculated for theparticular cutoff concentration, and 95% CI is the confidence intervalfor the odds ratio.

TABLE 1 Comparison of marker levels in urine samples collected fromCohort 1 (patients that did not progress beyond RIFLE stage 0) and inurine samples collected from subjects at 0, 24 hours, and 48 hours priorto reaching stage R, I or F in Cohort 2. Cancer Antigen 19-9 0 hr priorto AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage Cohort 1Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 14.7 27.514.7 29.5 14.7 37.5 Average 194 160 194 138 194 79.7 Stdev 1100 432 1100429 1100 127 p(t-test) 0.84 0.72 0.61 Min 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Max 11900 2270 11900 2940 11900 590 n (Samp) 12245 122 51 122 24 n (Patient) 99 45 99 51 99 24 sCr only Median 21.9 37.221.9 35.1 21.9 26.5 Average 168 68.7 168 122 168 97.2 Stdev 807 102 807213 807 172 p(t-test) 0.67 0.80 0.76 Min 1.00E−9 4.38 1.00E−9 3.941.00E−9 3.60 Max 11900 380 11900 758 11900 590 n (Samp) 259 12 259 19259 12 n (Patient) 159 12 159 19 159 12 UO only Median 15.5 27.5 15.527.9 15.5 30.3 Average 228 166 228 134 228 53.6 Stdev 1160 442 1160 4421160 57.8 p(t-test) 0.74 0.60 0.49 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 Max 11900 2270 11900 2940 11900 187 n (Samp) 109 43 10946 109 22 n (Patient) 85 43 85 46 85 22 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.58 0.58 0.550.60 0.61 0.55 0.60 0.57 0.54 SE 0.051 0.088 0.053 0.048 0.071 0.0510.066 0.088 0.069 p 0.12 0.34 0.34 0.038 0.13 0.30 0.12 0.43 0.53nCohort 1 122 259 109 122 259 109 122 259 109 nCohort 2 45 12 43 51 1946 24 12 22 Cutoff 1 13.1 13.7 13.4 17.3 17.3 17.5 12.5 18.5 9.76 Sens 171% 75% 72% 71% 74% 72% 71% 75% 73% Spec 1 48% 39% 44% 56% 44% 53% 47%46% 30% Cutoff 2 7.31 13.1 6.81 10.2 10.2 7.89 6.42 13.7 7.31 Sens 2 80%83% 81% 80% 84% 80% 83% 83% 82% Spec 2 26% 38% 22% 37% 32% 25% 25% 39%23% Cutoff 3 0 7.31 0 2.28 6.42 0.389 3.42 8.65 3.42 Sens 3 100%  92%100%  90% 95% 91% 92% 92% 91% Spec 3  0% 24%  0% 17% 22%  9% 18% 29% 13%Cutoff 4 31.3 57.3 53.0 31.3 57.3 53.0 31.3 57.3 53.0 Sens 4 47% 42% 30%49% 37% 28% 54% 25% 41% Spec 4 70% 70% 71% 70% 70% 71% 70% 70% 71%Cutoff 5 69.9 109 90.2 69.9 109 90.2 69.9 109 90.2 Sens 5 20%  8% 19%24% 16% 20% 25% 17% 23% Spec 5 80% 80% 81% 80% 80% 81% 80% 80% 81%Cutoff 6 215 289 504 215 289 504 215 289 504 Sens 6 13%  8%  7% 10% 11% 4%  8%  8%  0% Spec 6 90% 90% 91% 90% 90% 91% 90% 90% 91% OR Quart 20.97 0.98 0.42 0.86 2.0 0.51 0.55 4.1 0.74 p Value 0.95 0.99 0.16 0.790.42 0.24 0.43 0.21 0.68 95% CI of 0.34 0.13 0.13 0.30 0.36 0.16 0.120.45 0.18 OR Quart2 2.8 7.2 1.4 2.5 11 1.6 2.5 38 3.1 OR Quart 3 2.0 2.02.8 3.0 3.2 2.4 1.8 4.1 1.2 p Value 0.17 0.42 0.036 0.024 0.16 0.0730.36 0.21 0.78 95% CI of 0.75 0.36 1.1 1.2 0.62 0.92 0.52 0.45 0.33 ORQuart3 5.2 11 7.3 7.7 16 6.3 6.1 38 4.4 OR Quart 4 1.4 2.0 0.87 2.0 3.71.2 1.7 3.0 1.5 p Value 0.49 0.42 0.79 0.17 0.11 0.67 0.39 0.34 0.56 95%CI of 0.52 0.36 0.31 0.75 0.74 0.46 0.50 0.31 0.41 OR Quart4 3.9 11 2.55.1 19 3.4 5.8 30 5.2 C-C motif chemokine 13 0 hr prior to AKI stage 24hr prior to AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Average 2.84 4.72 2.84 5.68 2.84 4.14 Stdev 17.016.7 17.0 18.7 17.0 15.4 p(t-test) 0.38 0.16 0.63 Min 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 214 106 214 101 214 65.2 n (Samp)360 76 360 92 360 43 n (Patient) 190 76 190 92 190 43 sCr only Median1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Average 2.76 9.67 2.766.17 2.76 4.11 Stdev 15.7 32.6 15.7 17.9 15.7 12.8 p(t-test) 0.028 0.200.68 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 214 143 21486.2 214 51.0 n (Samp) 760 29 760 37 760 23 n (Patient) 297 29 297 37297 23 UO only Median 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9Average 2.93 5.27 2.93 6.34 2.93 4.38 Stdev 17.7 18.9 17.7 19.8 17.715.1 p(t-test) 0.34 0.14 0.64 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 Max 214 119 214 101 214 65.2 n (Samp) 317 66 317 79 31736 n (Patient) 136 66 136 79 136 36 0 hr prior to AKI stage 24 hr priorto AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr orUO sCr only UO only sCr or UO sCr only UO only AUC 0.53 0.54 0.53 0.540.55 0.54 0.50 0.53 0.52 SE 0.037 0.056 0.040 0.034 0.050 0.037 0.0470.062 0.051 p 0.41 0.50 0.39 0.30 0.33 0.25 1.00 0.60 0.67 nCohort 1 360760 317 360 760 317 360 760 317 nCohort 2 76 29 66 92 37 79 43 23 36Cutoff 1 0 0 0 0 0 0 0 0 0 Sens 1 100%  100%  100%  100%  100%  100% 100%  100%  100%  Spec 1  0%  0%  0%  0%  0%  0% 0%  0%  0% Cutoff 2 0 00 0 0 0 0 0 0 Sens 2 100%  100%  100%  100%  100%  100%  100%  100% 100%  Spec 2  0%  0%  0%  0%  0%  0% 0%  0%  0% Cutoff 3 0 0 0 0 0 0 0 00 Sens 3 100%  100%  100%  100%  100%  100%  100%  100%  100%  Spec 3 0%  0%  0%  0%  0%  0% 0%  0%  0% Cutoff 4 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Sens 4 13% 14% 14% 14%16% 15% 7% 13% 11% Spec 4 93% 93% 93% 93% 93% 93% 93%  93% 93% Cutoff 51.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9Sens 5 13% 14% 14% 14% 16% 15% 7% 13% 11% Spec 5 93% 93% 93% 93% 93% 93%93%  93% 93% Cutoff 6 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Sens 6 13% 14% 14% 14% 16% 15% 7% 13% 11% Spec 693% 93% 93% 93% 93% 93% 93%  93% 93% OR Quart 2 26 >29 39 41 >37 3230 >22 22 p Value 4.4E−11 <0.0011 3.4E−9  9.3E−14 <4.2E−4    7.5E−125.2E−6  <0.0026 3.5E−5  95% CI of 10.0 >3.8 12 15 >5.0 12 6.9 >2.9 5.1OR Quart2 70 na 130 110 na 85 130 na 97 OR Quart 3 0 >0 0 0 >0 0 0 >0 0p Value na <na   na na <na   na na <na   na 95% CI of na >na   nana >na   na na >na   na OR Quart3 na na na na na na na na na OR Quart 42.1 >4.1 3.2 2.8 >6.2 2.6 1.5 >3.0 2.0 p Value 0.19 <0.21 0.091 0.058<0.094 0.085 0.66 <0.34 0.42 95% CI of 0.69 >0.45 0.83 0.97 >0.73 0.880.25 >0.31 0.36 OR Quart4 6.4 na 12 8.2 na 7.7 9.2 na 11 C-X-C motifchemokine 6 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr priorto AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCror UO Median 13.2 24.1 13.2 19.1 13.2 8.72 Average 27.1 52.2 27.1 56.827.1 24.5 Stdev 59.3 169 59.3 127 59.3 34.7 p(t-test) 0.026 0.0012 0.78Min 1.00E−9 0.240 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 593 1450 593 769593 140 n (Samp) 358 75 358 92 358 43 n (Patient) 190 75 190 92 190 43sCr only Median 15.2 28.4 15.2 19.3 15.2 9.05 Average 39.5 117 39.5 85.739.5 22.7 Stdev 122 310 122 182 122 26.1 p(t-test) 0.0023 0.028 0.51 Min1.00E−9 0.240 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 2500 1450 2500 7692500 78.5 n (Samp) 758 29 758 37 758 23 n (Patient) 297 29 297 37 297 23UO only Median 13.9 28.7 13.9 21.4 13.9 10.0 Average 27.3 56.2 27.3 63.027.3 28.4 Stdev 56.5 180 56.5 136 56.5 39.2 p(t-test) 0.019 3.7E−4  0.91Min 1.00E−9 0.324 1.00E−9 0.324 1.00E−9 1.00E−9 Max 593 1450 593 769 593140 n (Samp) 315 65 315 79 315 36 n (Patient) 136 65 136 79 136 36 0 hrprior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCror UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UOonly AUC 0.59 0.55 0.62 0.59 0.57 0.59 0.46 0.44 0.46 SE 0.037 0.0560.040 0.034 0.050 0.037 0.047 0.063 0.052 p 0.014 0.38 0.0032 0.013 0.190.010 0.40 0.35 0.49 nCohort 1 358 758 315 358 758 315 358 758 315nCohort 2 75 29 65 92 37 79 43 23 36 Cutoff 1 7.26 5.99 9.02 7.68 9.6510.2 4.01 5.34 4.01 Sens 1 71% 72% 71% 71% 70% 71% 72% 74% 72% Spec 133% 23% 36% 35% 35% 38% 22% 22% 18% Cutoff 2 4.82 2.65 6.71 5.34 5.995.40 3.11 4.50 3.11 Sens 2 81% 83% 80% 80% 81% 82% 81% 83% 81% Spec 224% 11% 27% 28% 23% 23% 16% 18% 13% Cutoff 3 2.31 1.16 3.83 2.46 2.382.82 1.36 2.81 1.41 Sens 3 91% 93% 91% 90% 92% 91% 91% 91% 92% Spec 313%  6% 17% 14% 11% 12%  9% 12%  7% Cutoff 4 22.6 28.7 25.5 22.6 28.725.5 22.6 28.7 25.5 Sens 4 51% 48% 54% 45% 46% 42% 30% 30% 36% Spec 470% 70% 70% 70% 70% 70% 70% 70% 70% Cutoff 5 31.4 39.8 32.1 31.4 39.832.1 31.4 39.8 32.1 Sens 5 40% 38% 45% 34% 32% 35% 28% 26% 33% Spec 580% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6 51.6 64.0 53.8 51.6 64.053.8 51.6 64.0 53.8 Sens 6 20% 31% 15% 18% 22% 20% 16% 13% 14% Spec 690% 90% 90% 90% 90% 90% 90% 90% 90% OR Quart 2 0.86 0.43 0.83 1.1 0.870.66 0.14 0.33 0.066 p Value 0.69 0.17 0.66 0.88 0.78 0.30 0.011 0.180.0097 95% CI of 0.40 0.13 0.35 0.52 0.31 0.30 0.030 0.065 0.0085 ORQuart2 1.9 1.4 1.9 2.1 2.4 1.5 0.63 1.6 0.52 OR Quart 3 0.79 0.54 0.911.1 1.1 1.2 1.0 1.2 0.58 p Value 0.55 0.28 0.83 0.72 0.81 0.58 0.98 0.770.25 95% CI of 0.36 0.18 0.39 0.56 0.43 0.60 0.44 0.39 0.23 OR Quart31.7 1.6 2.1 2.3 3.0 2.5 2.3 3.6 1.5 OR Quart 4 2.4 1.2 2.8 2.3 1.7 2.11.2 1.4 1.1 p Value 0.011 0.66 0.0062 0.0094 0.27 0.035 0.66 0.58 0.8195% CI of 1.2 0.50 1.3 1.2 0.67 1.1 0.54 0.46 0.49 OR Quart4 4.7 3.0 5.84.5 4.1 4.1 2.7 4.0 2.5 Coagulation factor VII 0 hr prior to AKI stage24 hr prior to AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 2.63 4.77 2.63 5.052.63 3.72 Average 5.27 6.37 5.27 8.73 5.27 14.0 Stdev 8.53 5.43 8.5311.0 8.53 47.2 p(t-test) 0.39 0.0093 0.0097 Min 0.00408 0.00408 0.004080.00408 0.00408 0.00408 Max 65.5 20.4 65.5 63.6 65.5 249 n (Samp) 255 48255 57 255 27 n (Patient) 103 48 103 57 103 27 sCr only Median 3.35 2.463.35 6.36 3.35 3.86 Average 6.76 7.40 6.76 9.71 6.76 6.19 Stdev 14.412.1 14.4 10.0 14.4 6.27 p(t-test) 0.86 0.35 0.89 Min 0.00408 0.004080.00408 0.00408 0.00408 0.00408 Max 249 48.8 249 32.3 249 23.5 n (Samp)447 16 447 21 447 13 n (Patient) 170 16 170 21 170 13 UO only Median2.34 5.38 2.34 4.65 2.34 3.77 Average 4.84 6.59 4.84 8.10 4.84 14.0Stdev 8.24 5.17 8.24 10.8 8.24 49.1 p(t-test) 0.17 0.017 0.013 Min0.00408 0.409 0.00408 0.384 0.00408 0.303 Max 65.5 20.4 65.5 63.6 65.5249 n (Samp) 218 46 218 51 218 25 n (Patient) 87 46 87 51 87 25 0 hrprior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCror UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UOonly AUC 0.62 0.48 0.67 0.65 0.61 0.66 0.58 0.56 0.60 SE 0.046 0.0740.047 0.043 0.067 0.045 0.060 0.084 0.063 p 0.012 0.83 2.3E−4 5.7E−40.097 3.6E−4 0.16 0.49 0.10 nCohort 1 255 447 218 255 447 218 255 447218 nCohort 2 48 16 46 57 21 51 27 13 25 Cutoff 1 1.70 1.23 3.00 2.643.00 2.42 2.06 2.06 1.88 Sens 1 71% 75% 72% 72% 71% 71% 70% 77% 72% Spec1 39% 26% 57% 51% 47% 52% 43% 37% 46% Cutoff 2 1.24 0.952 1.72 2.09 2.421.79 1.70 1.81 1.67 Sens 2 81% 81% 80% 81% 81% 80% 81% 85% 80% Spec 231% 19% 43% 43% 41% 44% 39% 35% 41% Cutoff 3 0.564 0 0.704 0.906 0.6921.23 1.10 1.72 0.952 Sens 3 92% 100%  91% 91% 90% 90% 93% 92% 92% Spec 315%  0% 20% 22% 14% 32% 28% 33% 24% Cutoff 4 5.19 6.42 4.32 5.19 6.424.32 5.19 6.42 4.32 Sens 4 48% 38% 59% 47% 48% 53% 33% 38% 32% Spec 470% 70% 70% 70% 70% 70% 70% 70% 70% Cutoff 5 7.42 9.53 7.01 7.42 9.537.01 7.42 9.53 7.01 Sens 5 35% 19% 41% 35% 33% 35% 26% 23% 20% Spec 580% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6 11.5 15.3 10.1 11.5 15.310.1 11.5 15.3 10.1 Sens 6 19% 12% 24% 23% 24% 24% 11%  8% 16% Spec 690% 90% 90% 90% 90% 90% 90% 90% 90% OR Quart 2 1.1 0.74 1.4 1.6 1.3 2.44.9 5.2 1.7 p Value 0.82 0.70 0.55 0.34 0.73 0.12 0.046 0.14 0.48 95% CIof 0.41 0.16 0.44 0.61 0.33 0.80 1.0 0.60 0.39 OR Quart2 3.1 3.4 4.8 4.14.8 7.4 24 45 7.4 OR Quart 3 1.6 1.3 3.0 2.1 1.3 3.6 4.4 4.1 4.2 p Value0.36 0.73 0.050 0.12 0.73 0.020 0.068 0.21 0.035 95% CI of 0.60 0.33 1.00.83 0.33 1.2 0.90 0.45 1.1 OR Quart3 4.1 4.8 8.9 5.2 4.8 11 21 37 16 ORQuart 4 2.8 1.0 5.7 3.4 1.8 5.2 4.3 3.1 2.1 p Value 0.025 0.99 0.00120.0061 0.36 0.0022 0.071 0.34 0.32 95% CI of 1.1 0.25 2.0 1.4 0.51 1.80.88 0.31 0.49 OR Quart4 6.9 4.1 16 8.3 6.3 15 21 30 8.7 Insulin-likegrowth factor-binding protein 7 0 hr prior to AKI stage 24 hr prior toAKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 45.5 70.2 45.5 72.8 45.5 42.8 Average66.2 111 66.2 127 66.2 76.9 Stdev 69.6 104 69.6 176 69.6 76.2 p(t-test)5.0E−6 3.1E−7 0.35 Min 1.06 8.34 1.06 5.23 1.06 4.58 Max 533 594 5331250 533 382 n (Samp) 360 75 360 90 360 43 n (Patient) 190 75 190 90 19043 sCr only Median 51.8 61.7 51.8 106 51.8 80.7 Average 77.0 179 77.0167 77.0 94.5 Stdev 89.0 343 89.0 163 89.0 82.8 p(t-test) 9.0E−7 1.6E−80.35 Min 1.00E−9 8.34 1.00E−9 8.49 1.00E−9 4.58 Max 1250 1830 1250 6741250 344 n (Samp) 756 29 756 37 756 23 n (Patient) 296 29 296 37 296 23UO only Median 45.9 79.3 45.9 71.5 45.9 44.2 Average 67.4 120 67.4 13567.4 80.6 Stdev 68.6 103 68.6 187 68.6 80.9 p(t-test) 3.9E−7 3.8E−7 0.28Min 1.06 13.5 1.06 5.23 1.06 5.13 Max 533 594 533 1250 533 382 n (Samp)317 65 317 77 317 36 n (Patient) 136 65 136 77 136 36 0 hr prior to AKIstage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UO sCronly UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC0.67 0.61 0.71 0.65 0.71 0.64 0.54 0.59 0.54 SE 0.037 0.057 0.038 0.0340.049 0.037 0.047 0.063 0.052 p 3.0E−6 0.049 2.3E−8 1.7E−5 1.9E−5 9.5E−50.41 0.18 0.49 nCohort 1 360 756 317 360 756 317 360 756 317 nCohort 275 29 65 90 37 77 43 23 36 Cutoff 1 48.2 45.6 58.0 51.2 62.6 51.1 32.434.7 32.4 Sens 1 71% 72% 71% 70% 70% 70% 72% 74% 72% Spec 1 52% 44% 61%56% 59% 55% 36% 32% 33% Cutoff 2 37.2 24.8 45.6 28.5 49.1 28.5 23.0 29.923.0 Sens 2 80% 83% 80% 80% 81% 81% 81% 83% 81% Spec 2 41% 23% 49% 32%47% 29% 25% 28% 22% Cutoff 3 24.6 18.2 36.9 17.8 27.7 19.9 11.9 25.311.9 Sens 3 91% 93% 91% 90% 92% 91% 91% 91% 92% Spec 3 27% 16% 39% 18%26% 16%  9% 24%  7% Cutoff 4 69.8 79.3 73.1 69.8 79.3 73.1 69.8 79.373.1 Sens 4 52% 48% 52% 52% 59% 49% 40% 52% 39% Spec 4 70% 70% 70% 70%70% 70% 70% 70% 70% Cutoff 5 93.6 105 94.7 93.6 105 94.7 93.6 105 94.7Sens 5 40% 41% 46% 41% 51% 43% 33% 35% 33% Spec 5 80% 80% 80% 80% 80%80% 80% 80% 80% Cutoff 6 139 160 140 139 160 140 139 160 140 Sens 6 27%31% 32% 24% 30% 25% 14% 13% 14% Spec 6 90% 90% 90% 90% 90% 90% 90% 90%90% OR Quart 2 1.8 0.83 4.8 0.77 2.0 0.99 1.5 1.5 1.1 p Value 0.21 0.760.017 0.52 0.32 0.98 0.38 0.53 0.81 95% CI of 0.73 0.25 1.3 0.34 0.500.42 0.61 0.42 0.43 OR Quart2 4.2 2.8 17 1.7 8.2 2.3 3.7 5.4 2.9 ORQuart 3 2.6 1.0 7.7 2.0 3.1 1.8 0.64 0.74 0.42 p Value 0.023 1.0 0.00150.060 0.094 0.13 0.41 0.70 0.16 95% CI of 1.1 0.32 2.2 0.97 0.83 0.840.22 0.16 0.12 OR Quart3 6.0 3.2 27 3.9 12 4.0 1.9 3.4 1.4 OR Quart 44.2 2.1 14 3.1 6.9 3.6 1.8 2.6 1.5 p Value 4.7E−4 0.16 2.6E−5 8.1E−40.0022 6.6E−4 0.21 0.12 0.38 95% CI of 1.9 0.76 4.1 1.6 2.0 1.7 0.730.79 0.61 OR Quart4 9.3 5.6 48 6.2 24 7.5 4.2 8.3 3.7

TABLE 2 Comparison of marker levels in urine samples collected fromCohort 1 (patients that did not progress beyond RIFLE stage 0 or R) andin urine samples collected from subjects at 0, 24 hours, and 48 hoursprior to reaching stage I or F in Cohort 2. Cancer Antigen 19-9 0 hrprior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stageCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median21.9 38.2 21.9 28.5 21.9 17.7 Average 154 148 154 233 154 91.3 Stdev 810279 810 592 810 125 p(t-test) 0.97 0.60 0.76 Min 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Max 11900 1070 11900 2940 11900 371 n (Samp) 24122 241 32 241 16 n (Patient) 159 22 159 32 159 16 sCr only Median nd nd22.9 44.1 22.9 75.0 Average nd nd 160 46.8 160 140 Stdev nd nd 746 31.4746 225 p(t-test) nd nd 0.71 0.94 Min nd nd 1.00E−9 3.94 1.00E−9 3.60Max nd nd 11900 90.5 11900 645 n (Samp) nd nd 307 6 307 7 n (Patient) ndnd 186 6 186 7 UO only Median 23.1 38.2 23.1 29.3 23.1 34.8 Average 165149 165 252 165 95.1 Stdev 863 279 863 620 863 127 p(t-test) 0.93 0.600.76 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 11900 107011900 2940 11900 371 n (Samp) 210 22 210 29 210 15 n (Patient) 132 22132 29 132 15 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hrprior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO onlysCr or UO sCr only UO only AUC 0.59 nd 0.58 0.59 0.59 0.58 0.51 0.640.53 SE 0.066 nd 0.066 0.056 0.12 0.059 0.075 0.11 0.079 p 0.18 nd 0.250.097 0.48 0.15 0.87 0.22 0.67 nCohort 1 241 nd 210 241 307 210 241 307210 nCohort 2 22 nd 22 32 6 29 16 7 15 Cutoff 1 22.6 nd 22.6 20.7 26.620.7 6.23 57.7 9.76 Sens 1 73% nd 73% 72% 83% 72% 75% 71% 73% Spec 1 51%nd 48% 49% 55% 46% 20% 71% 28% Cutoff 2 6.42 nd 6.42 18.5 26.6 18.5 4.7614.2 6.23 Sens 2 82% nd 82% 81% 83% 83% 81% 86% 80% Spec 2 21% nd 19%47% 55% 45% 17% 38% 18% Cutoff 3 0 nd 0 3.42 3.42 1.00E−9 0 3.42 0 Sens3 100%  nd 100%  91% 100%  93% 100%  100%  100%  Spec 3  0% nd  0% 15%15%  5%  0% 15%  0% Cutoff 4 50.1 nd 54.2 50.1 57.3 54.2 50.1 57.3 54.2Sens 4 36% nd 36% 38% 33% 34% 44% 71% 40% Spec 4 70% nd 70% 70% 70% 70%70% 70% 70% Cutoff 5 86.3 nd 90.2 86.3 115 90.2 86.3 115 90.2 Sens 5 27%nd 27% 28%  0% 28% 38% 14% 33% Spec 5 80% nd 80% 80% 80% 80% 80% 80% 80%Cutoff 6 227 nd 239 227 289 239 227 289 239 Sens 6 18% nd 18% 16%  0%17% 12% 14% 13% Spec 6 90% nd 90% 90% 90% 90% 90% 90% 90% OR Quart 20.38 nd 0.38 1.4 0 2.1 0.79 0.99 0.74 p Value 0.25 nd 0.26 0.55 na 0.240.73 0.99 0.70 95% CI of 0.070 nd 0.070 0.44 na 0.60 0.20 0.061 0.16 ORQuart2 2.0 nd 2.0 4.8 na 7.4 3.1 16 3.4 OR Quart 3 1.7 nd 1.7 1.9 3.12.1 0.19 1.0 0.74 p Value 0.40 nd 0.38 0.27 0.33 0.24 0.13 1.0 0.70 95%CI of 0.51 nd 0.52 0.61 0.31 0.60 0.021 0.061 0.16 OR Quart3 5.4 nd 5.56.1 30 7.4 1.7 16 3.4 OR Quart 4 1.4 nd 1.5 2.4 2.0 2.4 1.2 4.1 1.2 pValue 0.56 nd 0.54 0.13 0.57 0.16 0.77 0.21 0.75 95% CI of 0.43 nd 0.430.78 0.18 0.70 0.35 0.45 0.32 OR Quart4 4.7 nd 4.9 7.3 23 8.4 4.1 38 4.9C-C motif chemokine 13 0 hr prior to AKI stage 24 hr prior to AKI stage48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1Cohort 2 sCr or UO Median 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 Average 2.76 7.45 2.76 9.69 2.76 3.39 Stdev 16.1 29.1 16.1 22.816.1 12.5 p(t-test) 0.10 0.0053 0.84 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 Max 214 143 214 101 214 61.9 n (Samp) 694 37 694 48 69428 n (Patient) 281 37 281 48 281 28 sCr only Median 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 Average 2.95 11.8 2.95 18.4 2.95 9.44Stdev 17.0 35.5 17.0 41.2 17.0 25.2 p(t-test) 0.13 0.0017 0.18 Min1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 241 106 241 143 24186.2 n (Samp) 904 9 904 13 904 13 n (Patient) 337 9 337 13 337 13 UOonly Median 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Average 2.934.84 2.93 10.6 2.93 3.80 Stdev 17.1 24.5 17.1 24.0 17.1 13.2 p(t-test)0.53 0.0058 0.80 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max214 143 214 101 214 61.9 n (Samp) 584 35 584 43 584 25 n (Patient) 20935 209 43 209 25 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hrprior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO onlysCr or UO sCr only UO only AUC 0.51 0.52 0.50 0.61 0.62 0.62 0.52 0.540.53 SE 0.049 0.098 0.050 0.044 0.084 0.047 0.056 0.083 0.060 p 0.860.81 0.95 0.012 0.15 0.012 0.73 0.59 0.65 nCohort 1 694 904 584 694 904584 694 904 584 nCohort 2 37 9 35 48 13 43 28 13 25 Cutoff 1 0 0 0 0 0 00 0 0 Sens 1 100%  100%  100%  100%  100%  100%  100%  100%  100%  Spec1 0%  0% 0%  0%  0%  0%  0%  0%  0% Cutoff 2 0 0 0 0 0 0 0 0 0 Sens 2100%  100%  100%  100%  100%  100%  100%  100%  100%  Spec 2 0%  0% 0% 0%  0%  0%  0%  0%  0% Cutoff 3 0 0 0 0 0 0 0 0 0 Sens 3 100%  100% 100%  100%  100%  100%  100%  100%  100%  Spec 3 0%  0% 0%  0%  0%  0% 0%  0%  0% Cutoff 4 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Sens 4 8% 11% 6% 29% 31% 30% 11% 15% 12% Spec 493%  93% 93%  93% 93% 93% 93% 93% 93% Cutoff 5 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Sens 5 8% 11% 6% 29% 31%30% 11% 15% 12% Spec 5 93%  93% 93%  93% 93% 93% 93% 93% 93% Cutoff 61.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9Sens 6 8% 11% 6% 29% 31% 30% 11% 15% 12% Spec 6 93%  93% 93%  93% 93%93% 93% 93% 93% OR Quart 2 >42 >8.3 0 >41 >9.4 35 >29 >12 >26 p Value<2.6E−4    <0.047 na <2.6E−4  <0.035 5.1E−4  <0.0010 <0.020 <0.0016 95%CI of >5.6 >1.0 na >5.6 >1.2 4.7 >3.9 >1.5 >3.4 OR Quart2 na na na na na260 na na na OR Quart 3 >0 >0 21 >0 >0 0 >0 >0 >0 p Value <na   <na  4.1E−5  <na   <na   na <na   <na   <na   95% CI of >na   >na  4.9 >na   >na   na >na   >na   >na   OR Quart3 na na 88 na na na na nana OR Quart 4 >3.0 >1.0 0 >15 >4.1 14 >3.0 >2.0 >3.0 p Value <0.34 <1.0na <0.0092 <0.21 0.012 <0.34 <0.57 <0.34 95% CI of >0.31 >0.062na >2.0 >0.45 1.8 >0.31 >0.18 >0.31 OR Quart4 na na na na na 110 na nana C-X-C motif chemokine 6 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 13.9 34.6 13.9 41.8 13.9 13.0 Average35.3 102 35.3 102 35.3 26.7 Stdev 119 279 119 172 119 35.5 p(t-test)0.0028 3.3E−4  0.70 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9Max 2500 1450 2500 769 2500 140 n (Samp) 693 37 693 48 693 28 n(Patient) 281 37 281 48 281 28 sCr only Median 15.6 66.7 15.6 51.3 15.615.0 Average 38.5 239 38.5 168 38.5 97.8 Stdev 114 466 114 300 114 209p(t-test) 9.4E−7  9.5E−5 0.066 Min 1.00E−9 4.70 1.00E−9  1.00E−9 1.00E−90.641 Max 2500 1450 2500 955 2500 769 n (Samp) 902 9 902 13 902 13 n(Patient) 337 9 337 13 337 13 UO only Median 14.9 28.4 14.9 40.8 14.921.2 Average 37.4 104 37.4 106 37.4 30.0 Stdev 128 287 128 180 128 37.1p(t-test) 0.0067 1.0E−3 0.77 Min 1.00E−9 1.00E−9 1.00E−9 1.65 1.00E−91.00E−9 Max 2500 1450 2500 769 2500 140 n (Samp) 583 35 583 43 583 25 n(Patient) 209 35 209 43 209 25 0 hr prior to AKI stage 24 hr prior toAKI stage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr or UOsCr only UO only sCr or UO sCr only UO only AUC 0.64 0.75 0.63 0.72 0.700.71 0.49 0.53 0.52 SE 0.050 0.095 0.052 0.043 0.082 0.045 0.056 0.0820.060 p 0.0042 0.0078 0.016 4.3E−7 0.017 3.1E−6 0.90 0.71 0.77 nCohort 1693 902 583 693 902 583 693 902 583 nCohort 2 37 9 35 48 13 43 28 13 25Cutoff 1 14.8 25.3 14.8 22.7 22.0 22.7 4.83 5.34 5.40 Sens 1 70% 78% 71%71% 77% 72% 71% 77% 72% Spec 1 53% 64% 50% 67% 60% 65% 21% 21% 22%Cutoff 2 7.91 7.91 11.8 9.65 6.00 9.65 3.84 4.01 4.82 Sens 2 81% 89% 80%81% 85% 81% 82% 85% 80% Spec 2 34% 30% 40% 39% 23% 35% 18% 16% 18%Cutoff 3 3.84 4.67 3.83 7.26 3.84 8.50 0.534 2.81 0.506 Sens 3 92% 100% 91% 92% 92% 91% 93% 92% 92% Spec 3 18% 18% 16% 30% 15% 33%  5% 12%  4%Cutoff 4 26.1 29.5 27.3 26.1 29.5 27.3 26.1 29.5 27.3 Sens 4 54% 67% 51%58% 69% 58% 36% 38% 36% Spec 4 70% 70% 70% 70% 70% 70% 70% 70% 70%Cutoff 5 35.9 41.2 36.6 35.9 41.2 36.6 35.9 41.2 36.6 Sens 5 46% 67% 40%54% 62% 53% 29% 38% 32% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80%Cutoff 6 58.1 64.7 60.3 58.1 64.7 60.3 58.1 64.7 60.3 Sens 6 22% 56% 17%38% 31% 37%  7% 31% 12% Spec 6 90% 90% 90% 90% 90% 90% 90% 90% 90% ORQuart 2 0.82 1.00 1.2 3.1 0 12 0.55 0.39 0.49 p Value 0.75 1.00 0.770.093 na 0.019 0.29 0.27 0.25 95% CI of 0.25 0.062 0.36 0.83 na 1.5 0.180.075 0.14 OR Quart2 2.7 16 4.0 12 na 92 1.7 2.0 1.7 OR Quart 3 1.2 1.001.4 3.1 0.66 7.3 0.43 0.20 0.49 p Value 0.78 1.00 0.56 0.093 0.65 0.0650.17 0.14 0.25 95% CI of 0.39 0.062 0.44 0.83 0.11 0.89 0.13 0.023 0.14OR Quart3 3.6 16 4.6 12 4.0 60 1.4 1.7 1.7 OR Quart 4 3.4 6.1 3.7 10 2.728 1.1 1.00 1.1 p Value 0.011 0.095 0.013 1.6E−4 0.14 0.0012 0.80 0.990.80 95% CI of 1.3 0.73 1.3 3.1 0.71 3.7 0.45 0.28 0.43 OR Quart4 8.7 5110 35 10 210 2.8 3.5 3.0 Coagulation factor VII 0 hr prior to AKI stage24 hr prior to AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 2.91 4.90 2.91 4.982.91 3.79 Average 6.32 5.78 6.32 7.80 6.32 4.26 Stdev 14.6 4.33 14.66.92 14.6 3.96 p(t-test) 0.85 0.56 0.56 Min 0.00408 0.423 0.00408 0.2620.00408 0.00408 Max 249 16.3 249 24.9 249 11.9 n (Samp) 421 25 421 33421 17 n (Patient) 165 25 165 33 165 17 sCr only Median nd nd 3.24 4.203.24 6.27 Average nd nd 6.60 9.15 6.60 6.89 Stdev nd nd 13.7 8.82 13.76.55 p(t-test) nd nd 0.60 0.96 Min nd nd 0.00408 2.19 0.00408 0.00408Max nd nd 249 24.9 249 19.4 n (Samp) nd nd 511 8 511 7 n (Patient) nd nd198 8 198 7 UO only Median 2.79 5.21 2.79 4.98 2.79 3.79 Average 6.146.06 6.14 7.51 6.14 4.39 Stdev 15.3 4.18 15.3 6.49 15.3 3.79 p(t-test)0.98 0.63 0.64 Min 0.00408 0.550 0.00408 0.262 0.00408 0.517 Max 24916.3 249 19.4 249 11.9 n (Samp) 357 25 357 29 357 17 n (Patient) 135 25135 29 135 17 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hrprior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO onlysCr or UO sCr only UO only AUC 0.60 nd 0.64 0.63 0.65 0.63 0.48 0.580.51 SE 0.061 nd 0.061 0.054 0.11 0.057 0.072 0.11 0.072 p 0.100 nd0.022 0.015 0.16 0.028 0.79 0.48 0.84 nCohort 1 421 nd 357 421 511 357421 511 357 nCohort 2 25 nd 25 33 8 29 17 7 17 Cutoff 1 3.28 nd 4.122.73 3.00 2.73 1.04 3.28 1.28 Sens 1 72% nd 72% 73% 75% 72% 71% 71% 71%Spec 1 53% nd 63% 48% 48% 50% 22% 50% 28% Cutoff 2 2.54 nd 3.28 2.162.42 1.43 0.837 1.81 0.906 Sens 2 80% nd 80% 82% 88% 83% 82% 86% 82%Spec 2 47% nd 55% 42% 42% 32% 18% 36% 19% Cutoff 3 0.762 nd 0.940 1.322.16 0.539 0.513 0 0.760 Sens 3 92% nd 92% 91% 100%  93% 94% 100%  94%Spec 3 17% nd 19% 30% 39% 11% 11%  0% 16% Cutoff 4 5.99 nd 5.61 5.996.42 5.61 5.99 6.42 5.61 Sens 4 36% nd 48% 45% 38% 48% 29% 43% 24% Spec4 70% nd 70% 70% 70% 70% 70% 70% 70% Cutoff 5 8.32 nd 8.12 8.32 9.318.12 8.32 9.31 8.12 Sens 5 16% nd 20% 36% 38% 41% 18% 29% 18% Spec 5 80%nd 80% 80% 80% 80% 80% 80% 80% Cutoff 6 13.6 nd 12.2 13.6 14.8 12.2 13.614.8 12.2 Sens 6  8% nd 12% 21% 25% 28%  0% 14%  0% Spec 6 90% nd 90%90% 90% 90% 90% 90% 90% OR Quart 2 0.74 nd 0.65 3.1 >4.1 1.5 1.3 0.990.58 p Value 0.69 nd 0.64 0.092 <0.21 0.53 0.72 1.00 0.47 95% CI of 0.16nd 0.11 0.83 >0.45 0.41 0.33 0.061 0.13 OR Quart2 3.4 nd 4.0 12 na 5.64.9 16 2.5 OR Quart 3 2.6 nd 4.4 2.4 >1.0 1.5 0.49 3.0 1.0 p Value 0.11nd 0.025 0.21 <1.0 0.52 0.42 0.34 1.0 95% CI of 0.80 nd 1.2 0.61 >0.0620.42 0.088 0.31 0.28 OR Quart3 8.7 nd 16 9.6 na 5.6 2.7 30 3.6 OR Quart4 2.1 nd 2.8 5.1 >3.0 3.6 1.5 2.0 0.78 p Value 0.25 nd 0.14 0.012 <0.340.032 0.51 0.57 0.72 95% CI of 0.60 nd 0.72 1.4 >0.31 1.1 0.42 0.18 0.20OR Quart4 7.0 nd 11 18 na 11 5.6 22 3.0 Insulin-like growthfactor-binding protein 7 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 49.6 73.9 49.6 121 49.6 76.1 Average70.0 177 70.0 203 70.0 114 Stdev 68.7 304 68.7 224 68.7 96.5 p(t-test) 5.5E−11 5.8E−23 0.0012 Min 1.00E−9 10.3 1.00E−9 13.1 1.00E−9 8.47 Max533 1830 533 1250 533 382 n (Samp) 691 37 691 48 691 28 n (Patient) 28037 280 48 280 28 sCr only Median 52.7 195 52.7 204 52.7 89.5 Average79.1 241 79.1 343 79.1 160 Stdev 90.5 170 90.5 473 90.5 144 p(t-test)1.5E−7 1.1E−18 0.0016 Min 1.00E−9 54.6 1.00E−9 17.1 1.00E−9 26.6 Max1250 594 1250 1830 1250 458 n (Samp) 900 9 900 13 900 13 n (Patient) 3369 336 13 336 13 UO only Median 50.1 73.9 50.1 119 50.1 85.5 Average 71.2173 71.2 202 71.2 122 Stdev 70.0 311 70.0 230 70.0 97.4 p(t-test) 9.2E−97.8E−19 5.1E−4 Min 1.00E−9 10.3 1.00E−9 13.1 1.00E−9 8.47 Max 533 1830533 1250 533 382 n (Samp) 581 35 581 43 581 25 n (Patient) 208 35 208 43208 25 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior toAKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr orUO sCr only UO only AUC 0.69 0.86 0.67 0.78 0.82 0.77 0.65 0.72 0.68 SE0.049 0.079 0.051 0.040 0.072 0.043 0.057 0.081 0.060 p 1.1E−4 6.1E−69.0E−4 2.2E−12 1.2E−5 5.3E−10 0.0088 0.0063 0.0035 nCohort 1 691 900 581691 900 581 691 900 581 nCohort 2 37 9 35 48 13 43 28 13 25 Cutoff 157.5 133 55.1 83.2 85.6 79.0 51.2 69.9 51.8 Sens 1 70% 78% 71% 71% 77%72% 71% 77% 72% Spec 1 58% 85% 55% 73% 72% 71% 51% 64% 52% Cutoff 2 46.568.6 39.6 65.8 73.9 65.8 34.6 54.9 47.0 Sens 2 81% 89% 80% 81% 85% 81%82% 85% 80% Spec 2 47% 62% 38% 63% 67% 63% 33% 52% 47% Cutoff 3 33.654.4 33.2 41.6 62.3 41.6 26.4 34.7 28.2 Sens 3 92% 100%  91% 92% 92% 91%93% 92% 92% Spec 3 32% 52% 30% 41% 58% 40% 26% 31% 27% Cutoff 4 76.282.2 77.2 76.2 82.2 77.2 76.2 82.2 77.2 Sens 4 49% 78% 49% 73% 77% 72%50% 54% 52% Spec 4 70% 70% 70% 70% 70% 70% 70% 70% 70% Cutoff 5 99.9 11299.6 99.9 112 99.6 99.9 112 99.6 Sens 5 49% 78% 46% 65% 69% 63% 36% 38%44% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6 146 163 147 146163 147 146 163 147 Sens 6 41% 56% 37% 40% 54% 37% 29% 38% 32% Spec 690% 90% 90% 90% 90% 90% 90% 90% 90% OR Quart 2 2.0 >0 2.4 1.7 0 1.0 3.11.0 2.5 p Value 0.32 <na   0.21 0.48 na 1.0 0.18 1.0 0.27 95% CI of0.50 >na   0.61 0.39 na 0.25 0.61 0.062 0.48 OR Quart2 8.3 na 9.4 7.1 na4.1 15 16 13 OR Quart 3 3.5 >2.0 2.8 2.7 3.0 1.8 4.1 5.1 3.1 p Value0.062 <0.57 0.14 0.14 0.34 0.36 0.076 0.14 0.17 95% CI of 0.94 >0.180.72 0.71 0.31 0.51 0.86 0.59 0.61 OR Quart3 13 na 11 10 29 6.2 20 44 16OR Quart 4 6.5 >7.2 6.2 13 9.3 8.3 6.3 6.1 6.4 p Value 0.0030 <0.0660.0040 3.6E−5  0.035 1.1E−4  0.017 0.095 0.016 95% CI of 1.9 >0.88 1.83.8 1.2 2.8 1.4 0.73 1.4 OR Quart4 23 na 22 42 74 24 29 51 29

TABLE 3 Comparison of marker levels in urine samples collected within 12hours of reaching stage R from Cohort 1 (patients that reached, but didnot progress beyond, RIFLE stage R) and from Cohort 2 (patients thatreached RIFLE stage I or F). Coagulation factor VII sCr or UO sCr onlyUO only Cohort Cohort Cohort Cohort Cohort Cohort 1 2 1 2 1 2 Median4.15 4.98 1.42 2.19 5.74 6.35 Average 5.72 6.64 3.27 4.92 6.37 8.23Stdev 5.41 6.33 3.88 5.72 5.35 6.91 p(t-test) 0.52 0.41 0.28 Min 0.004080.535 0.00408 0.842 0.409 0.423 Max 20.4 19.1 14.7 16.8 20.4 19.1 n(Samp) 52 23 18 7 43 16 n (Patient) 52 23 18 7 43 16 At Enrollment sCror UO sCr only UO only AUC 0.53 0.60 0.56 SE 0.073 0.13 0.086 p 0.660.43 0.48 nCohort 1 52 18 43 nCohort 2 23 7 16 Cutoff 1 1.45 1.45 2.87Sens 1 74% 71% 75% Spec 1 31% 56% 37% Cutoff 2 0.842 0.842 1.68 Sens 283% 86% 81% Spec 2 15% 22% 26% Cutoff 3 0.837 0.536 0.423 Sens 3 91%100%  94% Spec 3 15% 22%  2% Cutoff 4 8.08 4.03 8.49 Sens 4 30% 43% 38%Spec 4 71% 72% 72% Cutoff 5 10.3 6.57 10.9 Sens 5 26% 29% 38% Spec 5 81%83% 81% Cutoff 6 13.9 8.82 13.9 Sens 6 17% 14% 25% Spec 6 90% 94% 91% ORQuart 2 0.93 0 1.8 p Value 0.92 na 0.48 95% CI of 0.22 na 0.35 OR Quart24.0 na 9.7 OR Quart 3 1.5 1.0 0.56 p Value 0.56 1.0 0.57 95% CI of 0.380.091 0.079 OR Quart3 6.1 11 4.0 OR Quart 4 1.2 1.5 2.4 p Value 0.800.73 0.29 95% CI of 0.29 0.16 0.47 OR Quart4 4.9 14 13

TABLE 4 Comparison of the maximum marker levels in urine samplescollected from Cohort 1 (patients that did not progress beyond RIFLEstage 0) and the maximum values in urine samples collected from subjectsbetween enrollment and 0, 24 hours, and 48 hours prior to reaching stageF in Cohort 2. Cancer Antigen 19-9 0 hr prior to 24 hr prior to 48 hrprior to AKI stage AKI stage AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort2 Cohort 1 Cohort 2 sCr or UO Median 16.8 92.7 16.8 75.2 16.8 91.1Average 221 364 221 349 221 72.0 Stdev 1220 831 1220 835 1220 41.9 p(t-test) 0.69 0.72 0.75 Min 1.00E−9 6.68 1.00E−9 6.68 1.00E−9 16.2 Max11900 2940 11900 2940 11900 125 n (Samp) 99 12 99 12 99 7 n (Patient) 9912 99 12 99 7 sCr only Median 24.5 95.3 24.5 72.2 nd nd Average 230 184230 155 nd nd Stdev 1020 235 1020 242 nd nd p (t-test) 0.91 0.86 nd ndMin 1.00E−9 6.68 1.00E−9 6.68 nd nd Max 11900 645 11900 645 nd nd n(Samp) 159 6 159 6 nd nd n (Patient) 159 6 159 6 nd nd UO only Median18.5 96.9 18.5 96.9 18.5 76.8 Average 263 501 263 501 263 68.8 Stdev1310 1010 1310 1010 1310 45.0 p (t-test) 0.62 0.62 0.72 Min 1.00E−9 18.61.00E−9 18.6 1.00E−9 16.2 Max 11900 2940 11900 2940 11900 125 n (Samp)85 8 85 8 85 6 n (Patient) 85 8 85 8 85 6 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.69 0.760.73 0.64 0.76 0.73 nd 0.68 SE 0.085 0.12 0.10 0.086 0.12 0.10 0.11 nd0.12 p 0.0038 0.13 0.012 0.0079 0.25 0.012 0.036 nd 0.15 nCohort 1 99159 85 99 159 85 99 nd 85 nCohort 2 12 6 8 12 6 8 7 nd 6 Cutoff 1 50.150.8 57.4 33.3 33.3 57.4 57.4 nd 18.5 Sens 1 75% 83% 75% 75% 83% 75% 71%nd 83% Spec 1 73% 65% 72% 69% 59% 72% 75% nd 51% Cutoff 2 20.4 50.8 19.720.4 33.3 19.7 18.5 nd 18.5 Sens 2 83% 83% 88% 83% 83% 88% 86% nd 83%Spec 2 57% 65% 53% 57% 59% 53% 54% nd 51% Cutoff 3 18.5 6.42 18.5 18.56.42 18.5 16.1 nd 15.3 Sens 3 92% 100%  100%  92% 100%  100%  100%  nd100%  Spec 3 54% 18% 51% 54% 18% 51% 49% nd 46% Cutoff 4 43.0 66.9 55.343.0 66.9 55.3 43.0 nd 55.3 Sens 4 75% 67% 75% 67% 50% 75% 71% nd 67%Spec 4 71% 70% 71% 71% 70% 71% 71% nd 71% Cutoff 5 76.6 125 114 76.6 125114 76.6 nd 114 Sens 5 58% 33% 38% 50% 17% 38% 57% nd 17% Spec 5 81% 81%80% 81% 81% 80% 81% nd 80% Cutoff 6 227 396 504 227 396 504 227 nd 504Sens 6 17% 17% 25% 17% 17% 25%  0% nd  0% Spec 6 91% 91% 91% 91% 91% 91%91% nd 91% OR Quart 2 0.96 0 >1.0 0.96 0 >1.0 >1.0 nd >2.1 p Value 0.98na <0.98 0.98 na <0.98 <1.0 nd <0.56 95% CI of 0.057 na >0.062 0.057na >0.062 >0.059 nd >0.18 OR Quart 2 16 na na 16 na na na nd na OR Quart3 3.1 2.1 >2.2 4.3 2.1 >2.2 >2.2 nd >1.0 p Value 0.34 0.56 <0.53 0.200.56 <0.53 <0.54 nd <1.0 95% CI of 0.30 0.18 >0.18 0.45 0.18 >0.18 >0.18nd >0.059 OR Quart 3 32 24 na 42 24 na na nd na OR Quart 4 8.7 3.1 >6.17.1 3.1 >6.1 >4.5 nd >3.3 p Value 0.051 0.34 <0.11 0.080 0.34 <0.11<0.19 nd <0.32 95% CI of 0.99 0.31 >0.65 0.79 0.31 >0.65 >0.47 nd >0.32OR Quart 4 76 31 na 63 31 na na nd na C-C motif chemokine 13 0 hr priorto 24 hr prior to 48 hr prior to AKI stage AKI stage AKI stage Cohort 1Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Average 3.50 22.9 3.50 20.2 3.5011.9 Stdev 19.2 39.5 19.2 32.1 19.2 20.8 p (t-test)  1.6E−4  5.8E−4 0.16Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 214 143 214 101214 64.7 n (Samp) 190 21 190 21 190 11 n (Patient) 190 21 190 21 190 11sCr only Median 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Average5.03 18.9 5.03 9.33 5.03 1.17 Stdev 21.5 33.8 21.5 18.1 21.5 2.87 p(t-test) 0.041 0.51 0.66 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 Max 214 106 214 55.1 214 7.03 n (Samp) 297 11 297 11 297 6 n(Patient) 297 11 297 11 297 6 UO only Median 1.00E−9 7.03 1.00E−9 7.031.00E−9 7.03 Average 4.07 29.4 4.07 25.6 4.07 14.6 Stdev 21.9 44.8 21.936.0 21.9 22.2 p (t-test)  2.7E−4  1.0E−3 0.17 Min 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 214 143 214 101 214 64.7 n (Samp)136 15 136 15 136 9 n (Patient) 136 15 136 15 136 9 0 hr prior to AKIstage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UO sCronly UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC0.69 0.67 0.72 0.69 0.62 0.72 0.68 0.51 0.73 SE 0.066 0.091 0.077 0.0670.092 0.077 0.091 0.12 0.098 p 0.0034 0.067 0.0038 0.0035 0.20 0.00390.050 0.91 0.021 nCohort 1 190 297 136 190 297 136 190 297 136 nCohort 221 11 15 21 11 15 11 6 9 Cutoff 1 0 0 0 0 0 0 0 0 0 Sens 1 100%  100% 100%  100%  100%  100%  100%  100%  100%  Spec 1  0%  0%  0%  0%  0%  0% 0%  0%  0% Cutoff 2 0 0 0 0 0 0 0 0 0 Sens 2 100%  100%  100%  100% 100%  100%  100%  100%  100%  Spec 2  0%  0%  0%  0%  0%  0%  0%  0%  0%Cutoff 3 0 0 0 0 0 0 0 0 0 Sens 3 100%  100%  100%  100%  100%  100% 100%  100%  100%  Spec 3  0%  0%  0%  0%  0%  0%  0%  0%  0% Cutoff 41.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9Sens 4 48% 45% 53% 48% 36% 53% 45% 17% 56% Spec 4 90% 88% 90% 90% 88%90% 90% 88% 90% Cutoff 5 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Sens 5 48% 45% 53% 48% 36% 53% 45% 17% 56% Spec5 90% 88% 90% 90% 88% 90% 90% 88% 90% Cutoff 6 1.00E−9 7.03 4.08 1.00E−97.03 4.08 1.00E−9 7.03 4.08 Sens 6 48% 27% 53% 48% 18% 53% 45%  0% 56%Spec 6 90% 92% 90% 90% 92% 90% 90% 92% 90% OR Quart 2 12 5.3 >8.4 126.4 >8.4 5.4 4.1 >4.5 p Value 0.021 0.13 <0.053 0.021 0.089 <0.053 0.130.21 <0.19 95% CI of 1.5 0.60 >0.97 1.5 0.75 >0.97 0.61 0.45 >0.48 ORQuart 2 96 46 na 96 55 na 48 38 na OR Quart 3 0 0 >0 0 0 >0 0 0 >0 pValue na na <na na na <na na na <na 95% CI of na na >na na na >na nana >na OR Quart 3 na na na na na na na na na OR Quart 4 12 5.3 >9.9 124.2 >9.9 5.3 0.99 >5.6 p Value 0.021 0.13 <0.036 0.021 0.21 <0.036 0.130.99 <0.12 95% CI of 1.5 0.60 >1.2 1.5 0.45 >1.2 0.60 0.061 >0.62 ORQuart 4 96 46 na 96 38 na 47 16 na C—X—C motif chemokine 6 0 hr prior to24 hr prior to 48 hr prior to AKI stage AKI stage AKI stage Cohort 1Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 14.8 61.314.8 51.3 14.8 37.9 Average 34.0 241 34.0 163 34.0 64.9 Stdev 74.0 38274.0 232 74.0 66.9 p (t-test) 4.8E−10 7.8E−8 0.18 Min 1.00E−9 5.301.00E−9 1.62 1.00E−9 8.72 Max 593 1450 593 769 593 172 n (Samp) 190 21190 21 190 11 n (Patient) 190 21 190 21 190 11 sCr only Median 21.0 53.821.0 51.3 21.0 52.6 Average 57.2 253 57.2 171 57.2 182 Stdev 172 452 172282 172 292 p (t-test) 7.5E−4 0.036 0.083 Min 1.00E−9 5.30 1.00E−9 1.621.00E−9 17.6 Max 2500 1450 2500 769 2500 769 n (Samp) 297 11 297 11 2976 n (Patient) 297 11 297 11 297 6 UO only Median 17.8 163 17.8 114 17.824.7 Average 36.8 323 36.8 216 36.8 69.1 Stdev 76.2 428 76.2 256 76.274.0 p (t-test) 7.5E−11 6.3E−9 0.22 Min 1.00E−9 9.04 1.00E−9 9.041.00E−9 8.72 Max 593 1450 593 769 593 172 n (Samp) 136 15 136 15 136 9 n(Patient) 136 15 136 15 136 9 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.79 0.71 0.82 0.77 0.670.81 0.71 0.77 0.65 SE 0.060 0.089 0.068 0.062 0.091 0.069 0.090 0.110.10 p 1.3E−6 0.017 2.7E−6 1.8E−5 0.066 5.7E−6 0.018 0.016 0.14 nCohort1 190 297 136 190 297 136 190 297 136 nCohort 2 21 11 15 21 11 15 11 6 9Cutoff 1 33.0 33.0 47.5 24.0 22.0 47.5 17.6 37.6 14.3 Sens 1 71% 73% 73%71% 73% 73% 73% 83% 78% Spec 1 75% 64% 82% 66% 53% 82% 56% 69% 43%Cutoff 2 17.6 17.6 23.5 17.6 17.6 23.5 14.3 37.6 9.02 Sens 2 81% 82% 80%81% 82% 80% 82% 83% 89% Spec 2 56% 45% 61% 56% 45% 61% 49% 69% 30%Cutoff 3 11.5 11.5 11.9 11.5 11.5 11.9 9.03 17.6 8.41 Sens 3 90% 91% 93%90% 91% 93% 91% 100%  100%  Spec 3 43% 32% 38% 43% 32% 38% 36% 45% 29%Cutoff 4 27.0 38.6 30.7 27.0 38.6 30.7 27.0 38.6 30.7 Sens 4 71% 64% 73%67% 55% 73% 55% 67% 44% Spec 4 70% 70% 71% 70% 70% 71% 70% 70% 71%Cutoff 5 39.8 53.3 39.8 39.8 53.3 39.8 39.8 53.3 39.8 Sens 5 67% 55% 73%62% 45% 73% 45% 50% 44% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80%Cutoff 6 57.9 111 68.0 57.9 111 68.0 57.9 111 68.0 Sens 6 52% 36% 60%43% 27% 60% 27% 33% 33% Spec 6 90% 90% 90% 90% 90% 90% 90% 90% 90% ORQuart 2 3.1 2.0 >3.2 3.1 2.0 >3.2 >3.2 >1.0 >4.5 p Value 0.34 0.57 <0.330.34 0.57 <0.33 <0.32 <1.0 <0.19 95% CI of 0.31 0.18 >0.31 0.310.18 >0.31 >0.32 >0.061 >0.48 OR Quart 2 30 23 na 30 23 na na na na ORQuart 3 3.1 2.0 >1.0 4.2 2.0 >1.0 >2.1 >1.0 >1.0 p Value 0.34 0.57 <1.00.21 0.57 <1.0 <0.55 <1.0 <0.98 95% CI of 0.31 0.18 >0.060 0.450.18 >0.060 >0.18 >0.061 >0.062 OR Quart 3 30 23 na 39 23 na na na na ORQuart 4 18 6.4 >15 17 6.4 >15 >6.7 >4.2 >4.4 p Value 0.0059 0.089 <0.0120.0080 0.089 <0.012 <0.084 <0.21 <0.20 95% CI of 2.3 0.75 >1.8 2.10.75 >1.8 >0.77 >0.45 >0.46 OR Quart 4 150 55 na 130 55 na na na naCoagulation factor VII 0 hr prior to 24 hr prior to 48 hr prior to AKIstage AKI stage AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1Cohort 2 sCr or UO Median 4.12 7.44 4.12 7.41 4.12 5.89 Average 8.3312.6 8.33 10.3 8.33 8.14 Stdev 12.0 12.2 12.0 8.04 12.0 7.18 p (t-test)0.19 0.55 0.96 Min 0.00408 0.491 0.00408 0.348 0.00408 3.18 Max 65.548.8 65.5 24.9 65.5 24.8 n (Samp) 103 16 103 15 103 8 n (Patient) 103 16103 15 103 8 sCr only Median 5.99 13.4 5.99 13.2 nd nd Average 10.7 17.210.7 13.4 nd nd Stdev 21.3 15.8 21.3 9.62 nd nd p (t-test) 0.40 0.72 ndnd Min 0.00408 0.491 0.00408 0.348 nd nd Max 249 48.8 249 24.9 nd nd n(Samp) 170 8 170 8 nd nd n (Patient) 170 8 170 8 nd nd UO only Median3.83 7.44 3.83 7.41 3.83 4.54 Average 7.81 9.13 7.81 8.15 7.81 5.64Stdev 11.9 5.61 11.9 5.33 11.9 2.93 p (t-test) 0.73 0.93 0.66 Min0.00408 3.48 0.00408 3.18 0.00408 3.18 Max 65.5 19.4 65.5 19.4 65.5 10.7n (Samp) 87 10 87 9 87 6 n (Patient) 87 10 87 9 87 6 0 hr prior to AKIstage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UO sCronly UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC0.68 0.67 0.70 0.65 0.65 0.66 0.62 nd 0.58 SE 0.078 0.11 0.096 0.0810.11 0.10 0.11 nd 0.13 p 0.020 0.12 0.039 0.063 0.16 0.12 0.29 nd 0.51nCohort 1 103 170 87 103 170 87 103 nd 87 nCohort 2 16 8 10 15 8 9 8 nd6 Cutoff 1 5.28 6.42 5.28 5.28 6.42 3.72 3.72 nd 3.31 Sens 1 75% 75% 70%73% 75% 78% 75% nd 83% Spec 1 57% 54% 61% 57% 54% 49% 48% nd 46% Cutoff2 4.32 5.29 4.32 3.72 5.29 3.31 3.31 nd 3.31 Sens 2 81% 88% 80% 80% 88%89% 88% nd 83% Spec 2 53% 46% 56% 48% 46% 46% 43% nd 46% Cutoff 3 3.310.435 3.72 3.07 0.313 3.07 3.07 nd 3.07 Sens 3 94% 100%  90% 93% 100% 100%  100%  nd 100%  Spec 3 43%  4% 49% 40%  1% 43% 40% nd 43% Cutoff 47.83 10.3 7.42 7.83 10.3 7.42 7.83 nd 7.42 Sens 4 44% 50% 50% 40% 50%44% 25% nd 17% Spec 4 71% 71% 70% 71% 71% 70% 71% nd 70% Cutoff 5 12.814.3 10.1 12.8 14.3 10.1 12.8 nd 10.1 Sens 5 31% 50% 40% 27% 50% 33% 12%nd 17% Spec 5 81% 80% 80% 81% 80% 80% 81% nd 80% Cutoff 6 19.4 21.0 17.319.4 21.0 17.3 19.4 nd 17.3 Sens 6 19% 38% 10% 13% 25% 11% 12% nd  0%Spec 6 90% 90% 91% 90% 90% 91% 90% nd 91% OR Quart 2 3.1 0.98 >2.2 3.10.98 >3.4 >3.2 nd >3.4 p Value 0.34 0.99 <0.54 0.34 0.99 <0.30 <0.32 nd<0.30 95% CI of 0.30 0.059 >0.18 0.30 0.059 >0.33 >0.32 nd >0.33 ORQuart 2 32 16 na 32 16 na na nd na OR Quart 3 5.6 2.0 >4.8 5.82.0 >3.4 >3.2 nd >2.2 p Value 0.13 0.56 <0.18 0.12 0.56 <0.30 <0.32 nd<0.53 95% CI of 0.61 0.18 >0.50 0.64 0.18 >0.33 >0.32 nd >0.18 OR Quart3 51 23 na 53 23 na na nd na OR Quart 4 8.5 4.2 >4.6 7.0 4.2 >3.4 >2.1nd >1.0 p Value 0.053 0.21 <0.19 0.081 0.21 <0.30 <0.56 nd <1.0 95% CIof 0.98 0.45 >0.47 0.79 0.45 >0.33 >0.18 nd >0.059 OR Quart 4 74 39 na62 39 na na nd na Insulin-like growth factor-binding protein 7 0 hrprior to 24 hr prior to 48 hr prior to AKI stage AKI stage AKI stageCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median54.4 210 54.4 206 54.4 165 Average 77.4 379 77.4 297 77.4 173 Stdev 79.3440 79.3 285 79.3 92.7 p (t-test) 5.8E−15 1.8E−14 1.7E−4 Min 1.93 27.21.93 23.1 1.93 53.6 Max 533 1830 533 1250 533 382 n (Samp) 190 21 190 21190 11 n (Patient) 190 21 190 21 190 11 sCr only Median 74.0 210 74.0206 74.0 208 Average 109 314 109 215 109 232 Stdev 119 320 119 153 119141 p (t-test) 5.7E−7  0.0044 0.013 Min 1.93 27.2 1.93 23.1 1.93 53.6Max 1250 1120 1250 458 1250 458 n (Samp) 296 11 296 11 296 6 n (Patient)296 11 296 11 296 6 UO only Median 61.2 247 61.2 247 61.2 165 Average85.1 468 85.1 362 85.1 182 Stdev 81.9 490 81.9 308 81.9 93.3 p (t-test)5.3E−14 5.4E−14 8.9E−4 Min 1.93 80.7 1.93 80.7 1.93 80.7 Max 533 1830533 1250 533 382 n (Samp) 136 15 136 15 136 9 n (Patient) 136 15 136 15136 9 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior toAKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr orUO sCr only UO only AUC 0.85 0.74 0.91 0.84 0.72 0.91 0.84 0.80 0.85 SE0.054 0.087 0.051 0.055 0.089 0.052 0.076 0.11 0.082 p 1.2E−10 0.00651.3E−15 4.1E−10 0.013 2.9E−15 7.3E−6 0.0061 2.4E−5 nCohort 1 190 296 136190 296 136 190 296 136 nCohort 2 21 11 15 21 11 15 11 6 9 Cutoff 1 145145 163 140 140 163 103 145 103 Sens 1 71% 73% 73% 71% 73% 73% 73% 83%78% Spec 1 89% 78% 90% 88% 77% 90% 77% 78% 73% Cutoff 2 103 53.2 145 10353.2 145 100 145 100 Sens 2 81% 82% 80% 81% 82% 80% 82% 83% 89% Spec 277% 36% 87% 77% 36% 87% 75% 78% 71% Cutoff 3 53.2 39.0 103 53.2 39.0 10376.5 53.2 76.4 Sens 3 90% 91% 93% 90% 91% 93% 91% 100%  100%  Spec 3 49%24% 73% 49% 24% 73% 65% 36% 59% Cutoff 4 91.1 114 99.6 91.1 114 99.691.1 114 99.6 Sens 4 81% 73% 93% 81% 73% 93% 82% 83% 89% Spec 4 70% 70%71% 70% 70% 71% 70% 70% 71% Cutoff 5 114 154 127 114 154 127 114 154 127Sens 5 76% 64% 80% 76% 55% 80% 64% 67% 67% Spec 5 80% 80% 80% 80% 80%80% 80% 80% 80% Cutoff 6 151 257 172 151 257 172 151 257 172 Sens 6 67%36% 67% 62% 36% 67% 55% 33% 44% Spec 6 90% 90% 90% 90% 90% 90% 90% 90%90% OR Quart 2 2.0 0.49 >0 2.0 0.49 >0 >1.0 >1.0 >0 p Value 0.58 0.56<na 0.58 0.56 <na <0.99 <1.0 <na 95% CI of 0.18 0.043 >na 0.180.043 >na >0.062 >0.061 >na OR Quart 2 23 5.5 na 23 5.5 na na na na ORQuart 3 2.0 0 >3.2 2.0 0 >3.2 >3.2 >0 >3.3 p Value 0.58 na <0.33 0.58 na<0.33 <0.32 <na <0.31 95% CI of 0.18 na >0.31 0.18na >0.31 >0.32 >na >0.32 OR Quart 3 23 na na 23 na na na na na OR Quart4 22 4.3 >17 22 4.3 >17 >8.0 >5.3 >7.0 p Value 0.0033 0.072 <0.00810.0033 0.072 <0.0081 <0.057 <0.13 <0.080 95% CI of 2.8 0.88 >2.1 2.80.88 >2.1 >0.94 >0.60 >0.79 OR Quart 4 170 21 na 170 21 na na na na

TABLE 5 Comparison of marker levels in EDTA samples collected fromCohort 1 (patients that did not progress beyond RIFLE stage 0) and inEDTA samples collected from subjects at 0, 24 hours, and 48 hours priorto reaching stage R, I or F in Cohort 2. Cancer Antigen 19-9 0 hr priorto 24 hr prior to 48 hr prior to AKI stage AKI stage AKI stage Cohort 1Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 0.2040.311 0.204 0.316 0.204 0.205 Average 0.320 0.631 0.320 0.804 0.3200.486 Stdev 0.642 0.759 0.642 1.07 0.642 0.749 p (t-test) 0.023 0.00190.27 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 5.30 2.795.30 5.26 5.30 2.83 n (Samp) 81 37 81 45 81 26 n (Patient) 74 37 74 4574 26 sCr only Median 0.244 0.333 0.244 0.695 0.244 0.228 Average 0.4660.735 0.466 3.65 0.466 0.763 Stdev 0.757 0.824 0.757 10.9 0.757 1.08 p(t-test) 0.24  6.4E−5 0.26 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−91.00E−9 Max 5.30 2.77 5.30 41.6 5.30 2.83 n (Samp) 197 12 197 14 197 9 n(Patient) 131 12 131 14 131 9 UO only Median 0.244 0.311 0.244 0.2850.244 0.228 Average 0.484 0.563 0.484 0.738 0.484 0.334 Stdev 0.7740.703 0.774 1.05 0.774 0.432 p (t-test) 0.63 0.13 0.40 Min 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 5.30 2.79 5.30 5.26 5.301.94 n (Samp) 77 29 77 43 77 21 n (Patient) 64 29 64 43 64 21 0 hr priorto AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UOsCr only UO only sCr or UO sCr only UO only sCr or UO sCR only UO onlyAUC 0.65 0.62 0.55 0.67 0.69 0.58 0.53 0.54 0.45 SE 0.056 0.089 0.0640.052 0.081 0.055 0.066 0.10 0.072 p 0.0075 0.16 0.46 7.8E−4 0.018 0.160.62 0.69 0.48 nCohort 1 81 197 77 81 197 77 81 197 77 nCohort 2 37 1229 45 14 43 26 9 21 Cutoff 1 0.204 0.204 0.134 0.204 0.244 0.182 0.08510.134 0.0842 Sens 1 70% 75% 72% 71% 71% 72% 73% 78% 71% Spec 1 53% 43%29% 53% 52% 39% 28% 30% 22% Cutoff 2 0.124 0.198 0.0783 0.174 0.1750.134 0.0291 0.124 0.0291 Sens 2 81% 83% 83% 80% 93% 81% 85% 89% 86%Spec 2 35% 41% 14% 43% 36% 29% 15% 30% 10% Cutoff 3 1.00E−9 0.124 00.00741 0.175 1.00E−9 0 0 1.00E−9 Sens 3 92% 92% 100%  91% 93% 91% 100% 100%  90% Spec 3 10% 30%  0% 14% 36%  9%  0%  0%  9% Cutoff 4 0.3080.405 0.401 0.308 0.405 0.401 0.308 0.405 0.401 Sens 4 51% 50% 45% 51%57% 44% 42% 33% 29% Spec 4 70% 70% 70% 70% 70% 70% 70% 70% 70% Cutoff 50.357 0.590 0.633 0.357 0.590 0.633 0.357 0.590 0.633 Sens 5 49% 42% 24%49% 57% 30% 31% 33% 14% Spec 5 80% 81% 81% 80% 81% 81% 80% 81% 81%Cutoff 6 0.614 0.944 1.27 0.614 0.944 1.27 0.614 0.944 1.27 Sens 6 27%33% 14% 40% 29% 19% 19% 22%  5% Spec 6 90% 90% 91% 90% 90% 91% 90% 90%91% OR Quart 2 1.2 4.2 0.78 1.9 3.1 1.8 0.78 4.2 1.8 p Value 0.81 0.200.69 0.28 0.34 0.28 0.69 0.21 0.44 95% CI of 0.34 0.46 0.22 0.59 0.310.62 0.22 0.45 0.43 OR Quart 2 4.0 39 2.7 6.1 30 5.5 2.7 39 7.2 OR Quart3 1.5 2.0 1.0 1.7 2.0 1.4 0.47 1.0 1.3 p Value 0.54 0.57 1.0 0.38 0.580.57 0.28 1.0 0.71 95% CI of 0.43 0.18 0.29 0.52 0.18 0.45 0.12 0.0610.31 OR Quart 3 4.9 23 3.4 5.6 23 4.2 1.9 16 5.6 OR Quart 4 4.4 5.3 1.46.9 9.1 2.1 1.4 3.1 1.8 p Value 0.012 0.13 0.61 8.9E−4 0.041 0.18 0.610.34 0.44 95% CI of 1.4 0.60 0.42 2.2 1.1 0.71 0.42 0.31 0.43 OR Quart 414 47 4.4 22 75 6.2 4.4 30 7.2 C-C motif chemokine 13 0 hr prior to 24hr prior to 48 hr prior to AKI stage AKI stage AKI stage Cohort 1 Cohort2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 21.0 1.00E−9 21.022.6 21.0 49.4 Average 102 42.7 102 49.6 102 60.4 Stdev 364 79.8 36472.1 364 63.8 p (t-test) 0.39 0.41 0.64 Min 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Max 2520 361 2520 279 2520 284 n (Samp) 52 29 5234 52 18 n (Patient) 50 29 50 34 50 18 sCr only Median 8.21 54.8 8.2144.2 8.21 54.3 Average 58.9 92.4 58.9 101 58.9 76.7 Stdev 235 121 235180 235 87.9 p (t-test) 0.66 0.47 0.83 Min 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Max 2520 361 2520 713 2520 284 n (Samp) 132 10132 17 132 8 n (Patient) 100 10 100 17 100 8 UO only Median 21.0 1.00E−921.0 1.00E−9 21.0 38.0 Average 109 13.6 109 33.2 109 39.5 Stdev 362 21.9362 48.9 362 35.5 p (t-test) 0.21 0.26 0.49 Min 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Max 2520 62.8 2520 186 2520 96.3 n (Samp) 53 2353 30 53 13 n (Patient) 46 23 46 30 46 13 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCR only UO only AUC 0.44 0.63 0.320.49 0.59 0.43 0.64 0.71 0.54 SE 0.067 0.098 0.070 0.064 0.076 0.0660.079 0.11 0.091 p 0.36 0.19 0.010 0.92 0.22 0.28 0.076 0.043 0.69nCohort 1 52 132 53 52 132 53 52 132 53 nCohort 2 29 10 23 34 17 30 18 813 Cutoff 1 0 0 0 0 0 0 23.9 38.0 7.19 Sens 1 100%  100%  100%  100% 100%  100%  72% 75% 77% Spec 1  0%  0%  0%  0%  0%  0% 58% 64% 42%Cutoff 2 0 0 0 0 0 0 7.19 21.8 0 Sens 2 100%  100%  100%  100%  100% 100%  89% 88% 100%  Spec 2  0%  0%  0%  0%  0%  0% 44% 58%  0% Cutoff 30 0 0 0 0 0 0 0 0 Sens 3 100%  100%  100%  100%  100%  100%  100%  100% 100%  Spec 3  0%  0%  0%  0%  0%  0%  0%  0%  0% Cutoff 4 56.0 44.2 62.856.0 44.2 62.8 56.0 44.2 62.8 Sens 4 17% 60%  0% 29% 47% 20% 39% 62% 31%Spec 4 71% 72% 72% 71% 72% 72% 71% 72% 72% Cutoff 5 72.2 59.5 72.2 72.259.5 72.2 72.2 59.5 72.2 Sens 5 14% 40%  0% 21% 41% 17% 28% 38% 23% Spec5 81% 80% 81% 81% 80% 81% 81% 80% 81% Cutoff 6 105 92.0 105 105 92.0 105105 92.0 105 Sens 6 10% 40%  0% 15% 24% 13%  6% 12%  0% Spec 6 90% 90%91% 90% 90% 91% 90% 90% 91% OR Quart 2 1.3 0 3.0 0.60 >10 1.2 4.6 0 4.6p Value 0.66 na 0.22 0.42 <0.033 0.74 0.20 na 0.20 95% CI of 0.36 na0.51 0.17 >1.2 0.34 0.46 na 0.46 OR Quart 2 5.0 na 18 2.1 na 4.6 46 na47 OR Quart 3 2.0 0.23 9.4 1.0 >1.0 2.3 8.7 4.4 5.0 p Value 0.28 0.200.010 1.0 <0.98 0.21 0.059 0.20 0.17 95% CI of 0.56 0.024 1.70.31 >0.062 0.63 0.92 0.46 0.49 OR Quart 3 7.5 2.2 53 3.3 na 8.1 83 4151 OR Quart 4 1.3 1.2 3.9 0.60 >9.9 1.3 10 3.2 4.6 p Value 0.66 0.760.13 0.42 <0.036 0.66 0.042 0.33 0.20 95% CI of 0.36 0.31 0.68 0.17 >1.20.36 1.1 0.32 0.46 OR Quart 4 5.0 5.1 23 2.1 na 5.0 95 32 47 C—X—C motifchemokine 6 0 hr prior to 24 hr prior to 48 hr prior to AKI stage AKIstage AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2sCr or UO Median 31.8 31.0 31.8 36.4 31.8 17.0 Average 49.0 49.8 49.051.5 49.0 32.9 Stdev 48.9 51.8 48.9 61.6 48.9 36.7 p (t-test) 0.95 0.840.21 Min 5.55 3.49 5.55 4.02 5.55 6.54 Max 244 244 244 311 244 144 n(Samp) 53 29 53 34 53 18 n (Patient) 51 29 51 34 51 18 sCr only Median26.4 31.9 26.4 44.1 26.4 28.4 Average 45.1 46.7 45.1 70.0 45.1 47.5Stdev 52.8 41.5 52.8 79.0 52.8 48.3 p (t-test) 0.92 0.087 0.90 Min 3.4910.7 3.49 8.88 3.49 9.55 Max 311 129 311 336 311 144 n (Samp) 133 10 13317 133 8 n (Patient) 101 10 101 17 101 8 UO only Median 31.3 29.1 31.336.4 31.3 19.6 Average 45.1 46.5 45.1 52.1 45.1 25.7 Stdev 46.6 53.446.6 65.3 46.6 19.2 p (t-test) 0.91 0.57 0.15 Min 5.55 3.49 5.55 4.025.55 6.54 Max 244 244 244 311 244 66.2 n (Samp) 54 23 54 30 54 13 n(Patient) 47 23 47 30 47 13 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCR only UO only AUC 0.48 0.54 0.48 0.48 0.630.50 0.32 0.50 0.34 SE 0.067 0.097 0.073 0.064 0.076 0.066 0.078 0.110.090 P 0.79 0.70 0.75 0.81 0.076 0.96 0.024 0.98 0.081 nCohort 1 53 13354 53 133 54 53 133 54 nCohort 2 29 10 23 34 17 30 18 8 13 Cutoff 1 17.626.2 14.8 17.9 31.5 17.6 12.2 13.9 10.7 Sens 1 72% 70% 74% 71% 71% 70%72% 75% 77% Spec 1 26% 50% 15% 26% 56% 28%  8% 20% 11% Cutoff 2 11.013.1 10.7 15.6 17.9 13.4 9.82 11.5 9.82 Sens 2 83% 80% 83% 82% 82% 80%83% 88% 85% Spec 2  8% 16% 11% 11% 34% 13%  8% 14%  9% Cutoff 3 8.8810.7 6.26 6.68 9.95 6.68 8.88 8.88 8.88 Sens 3 93% 90% 91% 91% 94% 90%94% 100%  92% Spec 3  8% 14%  2%  2% 11%  2%  8% 11%  9% Cutoff 4 46.943.7 43.7 46.9 43.7 43.7 46.9 43.7 43.7 Sens 4 34% 30% 30% 32% 53% 37%22% 38% 15% Spec 4 72% 71% 70% 72% 71% 70% 72% 71% 70% Cutoff 5 56.760.9 54.5 56.7 60.9 54.5 56.7 60.9 54.5 Sens 5 31% 30% 26% 26% 41% 23%22% 25% 15% Spec 5 81% 80% 81% 81% 80% 81% 81% 80% 81% Cutoff 6 117 95.294.9 117 95.2 94.9 117 95.2 94.9 Sens 6 10% 20%  9%  9% 18% 10%  6% 12% 0% Spec 6 91% 90% 91% 91% 90% 91% 91% 90% 91% OR Quart 2 0.44 0.30 0.501.0 0.63 1.0 0.21 1.0 1.0 p Value 0.23 0.31 0.34 1.0 0.62 1.0 0.18 0.981.0 95% CI of 0.12 0.030 0.12 0.30 0.099 0.29 0.021 0.14 0.12 OR Quart 21.7 3.1 2.1 3.3 4.0 3.5 2.1 7.7 8.1 OR Quart 3 0.67 0.97 0.66 0.67 1.80.65 1.3 0.50 2.3 p Value 0.53 0.97 0.56 0.53 0.46 0.51 0.70 0.58 0.3895% CI of 0.19 0.18 0.17 0.20 0.39 0.18 0.30 0.043 0.36 OR Quart 3 2.35.2 2.6 2.3 8.0 2.4 6.1 5.8 15 OR Quart 4 0.89 0.97 1.1 1.1 2.6 1.0 3.11.6 3.4 p Value 0.85 0.97 0.90 0.90 0.20 1.0 0.13 0.62 0.19 95% CI of0.26 0.18 0.29 0.32 0.61 0.29 0.72 0.25 0.56 OR Quart 4 3.1 5.2 4.0 3.611 3.5 13 10 21 Coagulation factor VII 0 hr prior to 24 hr prior to 48hr prior to AKI stage AKI stage AKI stage Cohort 1 Cohort 2 Cohort 1Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 312 313 312 280 312 290Average 339 323 339 283 339 310 Stdev 159 179 159 165 159 165 p (t-test)0.52 0.017 0.37 Min 2.08 22.9 2.08 12.2 2.08 66.4 Max 743 759 743 717743 685 n (Samp) 262 51 262 56 262 26 n (Patient) 110 51 110 56 110 26sCr only Median 298 336 298 324 298 418 Average 313 356 313 361 313 391Stdev 164 187 164 219 164 206 p (t-test) 0.28 0.20 0.095 Min 2.08 1082.08 66.2 2.08 62.4 Max 822 759 822 779 822 685 n (Samp) 466 18 466 21466 13 n (Patient) 180 18 180 21 180 13 UO only Median 310 296 310 262310 261 Average 335 304 335 256 335 271 Stdev 160 174 160 144 160 119 p(t-test) 0.21 0.0012 0.063 Min 16.0 22.9 16.0 12.2 16.0 66.4 Max 1020722 1020 574 1020 488 n (Samp) 221 50 221 52 221 23 n (Patient) 91 50 9152 91 23 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior toAKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr orUO sCR only UO only AUC 0.47 0.56 0.45 0.40 0.55 0.36 0.44 0.62 0.40 SE0.045 0.071 0.046 0.043 0.066 0.045 0.061 0.084 0.065 p 0.43 0.40 0.230.017 0.45 0.0022 0.36 0.17 0.11 nCohort 1 262 466 221 262 466 221 262466 221 nCohort 2 51 18 50 56 21 52 26 13 23 Cutoff 1 193 225 176 165190 156 220 187 220 Sens 1 71% 72% 72% 71% 71% 71% 73% 77% 74% Spec 118% 35% 14% 12% 27% 11% 26% 26% 27% Cutoff 2 140 182 138 138 138 100 134137 129 Sens 2 80% 83% 80% 80% 81% 81% 81% 85% 83% Spec 2  8% 24%  8% 8% 14%  4%  7% 13%  6% Cutoff 3 104 126 77.8 80.6 108 76.2 116 124 116Sens 3 90% 94% 90% 91% 90% 90% 92% 92% 91% Spec 3  4% 12%  3%  3%  8% 3%  5% 11%  5% Cutoff 4 411 394 393 411 394 393 411 394 393 Sens 4 29%39% 34% 23% 43% 21% 27% 62% 17% Spec 4 70% 70% 70% 70% 70% 70% 70% 70%70% Cutoff 5 481 459 479 481 459 479 481 459 479 Sens 5 22% 28% 18% 12%38%  8% 15% 38%  4% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6571 535 550 571 535 550 571 535 550 Sens 6 10% 22% 10%  5% 24%  2%  8%31%  0% Spec 6 90% 90% 90% 90% 90% 90% 90% 90% 90% OR Quart 2 1.2 1.01.2 0.91 0.48 1.3 1.2 0.32 2.1 p Value 0.64 1.0 0.65 0.84 0.31 0.60 0.750.33 0.31 95% CI of 0.53 0.24 0.51 0.36 0.12 0.50 0.35 0.033 0.50 ORQuart 2 2.8 4.1 3.0 2.3 2.0 3.3 4.2 3.2 8.9 OR Quart 3 0.64 1.0 0.69 1.10.48 1.1 1.7 1.3 2.9 p Value 0.36 1.0 0.46 0.82 0.31 0.78 0.39 0.71 0.1395% CI of 0.25 0.24 0.26 0.46 0.12 0.44 0.52 0.29 0.74 OR Quart 3 1.74.1 1.8 2.7 2.0 3.0 5.4 6.1 12 OR Quart 4 1.6 1.5 1.9 2.6 1.5 3.2 1.41.7 2.1 p Value 0.29 0.52 0.13 0.020 0.44 0.0086 0.55 0.48 0.31 95% CIof 0.69 0.42 0.82 1.2 0.53 1.3 0.44 0.39 0.50 OR Quart 4 3.5 5.5 4.4 5.74.4 7.6 4.8 7.2 8.9 Insulin-like growth factor-binding protein 7 0 hrprior to 24 hr prior to 48 hr prior to AKI stage AKI stage AKI stageCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median63.1 74.8 63.1 82.4 63.1 75.2 Average 68.7 77.4 68.7 94.1 68.7 74.1Stdev 34.7 31.0 34.7 68.0 34.7 20.5 p (t-test) 0.19 0.0063 0.46 Min 18.635.4 18.6 30.8 18.6 28.3 Max 250 185 250 437 250 114 n (Samp) 81 37 8145 81 26 n (Patient) 74 37 74 45 74 26 sCr only Median 67.7 75.4 67.792.0 67.7 74.4 Average 75.5 78.1 75.5 113 75.5 74.4 Stdev 41.1 39.2 41.159.9 41.1 25.4 p (t-test) 0.83 0.0017 0.94 Min 18.6 35.4 18.6 30.8 18.628.3 Max 437 185 437 233 437 116 n (Samp) 197 12 197 14 197 9 n(Patient) 131 12 131 14 131 9 UO only Median 69.3 76.7 69.3 81.8 69.377.9 Average 74.2 80.9 74.2 92.0 74.2 82.1 Stdev 33.6 32.7 33.6 65.133.6 30.6 p (t-test) 0.36 0.051 0.33 Min 27.8 39.1 27.8 36.0 27.8 39.8Max 250 189 250 437 250 185 n (Samp) 77 29 77 43 77 21 n (Patient) 64 2964 43 64 21 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr priorto AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCror UO sCR only UO only AUC 0.61 0.52 0.58 0.65 0.72 0.60 0.62 0.54 0.61SE 0.057 0.087 0.064 0.052 0.079 0.055 0.066 0.10 0.072 P 0.064 0.830.23 0.0039 0.0049 0.081 0.071 0.71 0.14 nCohort 1 81 197 77 81 197 7781 197 77 nCohort 2 37 12 29 45 14 43 26 9 21 Cutoff 1 56.1 68.2 56.556.3 82.4 53.8 58.3 69.0 62.3 Sens 1 70% 75% 72% 71% 71% 72% 73% 78% 71%Spec 1 44% 51% 36% 44% 66% 31% 47% 51% 42% Cutoff 2 53.1 39.8 53.3 53.170.5 52.5 56.1 49.8 58.1 Sens 2 81% 83% 83% 80% 86% 81% 81% 89% 81% Spec2 40% 10% 31% 40% 53% 27% 44% 22% 39% Cutoff 3 39.4 36.7 47.5 42.8 56.542.8 49.8 27.8 53.3 Sens 3 92% 92% 93% 91% 93% 91% 92% 100%  90% Spec 316%  6% 17% 20% 38% 12% 32%  2% 31% Cutoff 4 77.3 85.8 84.2 77.3 85.884.2 77.3 85.8 84.2 Sens 4 46% 17% 38% 53% 64% 49% 42% 22% 38% Spec 470% 70% 70% 70% 70% 70% 70% 70% 70% Cutoff 5 85.8 96.3 87.7 85.8 96.387.7 85.8 96.3 87.7 Sens 5 30% 17% 31% 47% 43% 44% 27% 22% 38% Spec 580% 80% 81% 80% 80% 81% 80% 80% 81% Cutoff 6 107 116 114 107 116 114 107116 114 Sens 6 11%  8% 14% 18% 29% 19%  4% 11% 10% Spec 6 90% 90% 91%90% 90% 91% 90% 90% 91% OR Quart 2 1.4 0.32 3.2 2.5 0.98 1.2 4.2 0 7.3 pValue 0.59 0.33 0.082 0.12 0.99 0.78 0.094 na 0.078 95% CI of 0.42 0.0320.86 0.80 0.060 0.39 0.78 na 0.80 OR Quart 2 4.7 3.2 12 7.8 16 3.5 23 na66 OR Quart 3 2.7 2.1 1.6 1.4 5.3 0.71 6.0 2.7 7.7 p Value 0.094 0.300.48 0.54 0.13 0.56 0.033 0.26 0.070 95% CI of 0.85 0.50 0.41 0.44 0.600.22 1.2 0.49 0.85 OR Quart 3 8.7 9.0 6.7 4.8 47 2.2 31 14 70 OR Quart 42.2 0.64 2.8 6.1 7.8 3.1 5.1 0.98 11 p Value 0.18 0.63 0.14 0.0018 0.0600.040 0.056 0.98 0.032 95% CI of 0.69 0.10 0.73 2.0 0.92 1.1 0.96 0.131.2 OR Quart 4 7.1 4.0 10 19 65 8.8 27 7.2 95

TABLE 6 Comparison of marker levels in EDTA samples collected fromCohort 1 (patients that did not progress beyond RIFLE stage 0 or R) andin EDTA samples collected from subjects at 0, 24 hours, and 48 hoursprior to reaching stage I or F in Cohort 2. Cancer Antigen 19-9 24 hrprior to 48 hr prior to 0 hr prior to AKI stage AKI stage AKI stageCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median0.228 0.629 0.228 0.536 0.228 0.489 Average 0.628 1.13 0.628 0.885 0.6280.805 Stdev 3.00 1.07 3.00 1.17 3.00 1.05 p(t-test) 0.66 0.70 0.81 Min1.00E−9 0.231 1.00E−9 1.00E−9 1.00E−9 0.0291 Max 41.6 2.77 41.6 5.2641.6 3.84 n (Samp) 197 7 197 21 197 16 n (Patient) 131 7 131 21 131 16UO only Median nd nd 0.244 0.467 0.244 0.568 Average nd nd 0.695 0.8310.695 0.776 Stdev nd nd 3.19 1.21 3.19 1.04 p (t-test) nd nd 0.86 0.93Min nd nd 1.00E−9 1.00E−9 1.00E−9 0.0291 Max nd nd 41.6 5.26 41.6 3.84 n(Samp) nd nd 173 18 173 13 n (Patient) nd nd 111 18 111 13 0 hr prior toAKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UOsCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO onlyAUC 0.79 nd nd 0.71 nd 0.66 0.63 nd 0.62 SE 0.10 nd nd 0.066 nd 0.0730.077 nd 0.086 p 0.0047 nd nd 0.0013 nd 0.031 0.081 nd 0.15 nCohort 1197 nd nd 197 nd 173 197 nd 173 nCohort 2 7 nd nd 21 nd 18 16 nd 13Cutoff 1 0.401 nd nd 0.259 nd 0.257 0.204 nd 0.204 Sens 1 71% nd nd 71%nd 72% 75% nd 77% Spec 1 71% nd nd 55% nd 53% 44% nd 41% Cutoff 2 0.288nd nd 0.250 nd 0.228 0.134 nd 0.0842 Sens 2 86% nd nd 81% nd 83% 81% nd85% Spec 2 61% nd nd 55% nd 50% 30% nd 22% Cutoff 3 0.228 nd nd 0.174 nd0.134 0.0783 nd 0.0783 Sens 3 100%  nd nd 90% nd 94% 94% nd 92% Spec 352% nd nd 36% nd 29% 20% nd 18% Cutoff 4 0.361 nd nd 0.361 nd 0.4560.361 nd 0.456 Sens 4 71% nd nd 67% nd 50% 56% nd 54% Spec 4 70% nd nd70% nd 71% 70% nd 71% Cutoff 5 0.590 nd nd 0.590 nd 0.633 0.590 nd 0.633Sens 5 57% nd nd 43% nd 33% 38% nd 23% Spec 5 80% nd nd 80% nd 80% 80%nd 80% Cutoff 6 0.980 nd nd 0.980 nd 1.32 0.980 nd 1.32 Sens 6 43% nd nd24% nd 11% 19% nd 15% Spec 6 90% nd nd 90% nd 90% 90% nd 90% OR Quart2 >0 nd nd 3.1 nd 3.1 0.65 nd 0.31 p Value <na nd nd 0.34 nd 0.34 0.65nd 0.32 95% CI of >na nd nd 0.31 nd 0.31 0.10 nd 0.031 OR Quart 2 na ndnd 30 nd 31 4.1 nd 3.1 OR Quart 3 >3.2 nd nd 6.6 nd 7.9 1.0 nd 1.0 pValue <0.32 nd nd 0.085 nd 0.059 1.0 nd 1.0 95% CI of >0.32 nd nd 0.77nd 0.93 0.19 nd 0.19 OR Quart 3 na nd nd 57 nd 67 5.2 nd 5.2 OR Quart4 >4.3 nd nd 13 nd 7.9 2.9 nd 2.1 p Value <0.20 nd nd 0.015 nd 0.0590.13 nd 0.32 95% CI of >0.47 nd nd 1.6 nd 0.93 0.72 nd 0.49 OR Quart 4na nd nd 110 nd 67 12 nd 8.9 C-C motif chemokine 13 24 hr prior to 48 hrprior to 0 hr prior to AKI stage AKI stage AKI stage Cohort 1 Cohort 2Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 21.0 21.8 21.01.00E−9 21.0 44.5 Average 66.3 56.7 66.3 29.4 66.3 90.1 Stdev 235 86.8235 61.3 235 140 p (t-test) 0.92 0.48 0.72 Min 1.00E−9 1.00E−9 1.00E−91.00E−9 1.00E−9 1.00E−9 Max 2520 238 2520 279 2520 484 n (Samp) 130 7130 21 130 13 n (Patient) 102 7 102 21 102 13 UO only Median nd nd 21.01.00E−9 21.0 44.5 Average nd nd 70.0 16.9 70.0 74.5 Stdev nd nd 253 20.5253 140 p (t-test) nd nd 0.34 0.95 Min nd nd 1.00E−9 1.00E−9 1.00E−91.00E−9 Max nd nd 2520 44.5 2520 484 n (Samp) nd nd 112 21 112 11 n(Patient) nd nd 87 21 87 11 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.51 nd nd 0.40 nd 0.39 0.59nd 0.54 SE 0.11 nd nd 0.070 nd 0.070 0.087 nd 0.093 p 0.91 nd nd 0.14 nd0.11 0.28 nd 0.70 nCohort 1 130 nd nd 130 nd 112 130 nd 112 nCohort 2 7nd nd 21 nd 21 13 nd 11 Cutoff 1 0 nd nd 0 nd 0 0 nd 0 Sens 1 100%  ndnd 100%  nd 100%  100%  nd 100%  Spec 1  0% nd nd  0% nd  0%  0% nd  0%Cutoff 2 0 nd nd 0 nd 0 0 nd 0 Sens 2 100%  nd nd 100%  nd 100%  100% nd 100%  Spec 2  0% nd nd  0% nd  0%  0% nd  0% Cutoff 3 0 nd nd 0 nd 00 nd 0 Sens 3 100%  nd nd 100%  nd 100%  100%  nd 100%  Spec 3  0% nd nd 0% nd  0%  0% nd  0% Cutoff 4 56.0 nd nd 56.0 nd 56.0 56.0 nd 56.0 Sens4 29% nd nd 10% nd  0% 38% nd 36% Spec 4 73% nd nd 73% nd 72% 73% nd 72%Cutoff 5 72.2 nd nd 72.2 nd 72.2 72.2 nd 72.2 Sens 5 29% nd nd  5% nd 0% 31% nd 27% Spec 5 82% nd nd 82% nd 81% 82% nd 81% Cutoff 6 111 nd nd111 nd 111 111 nd 111 Sens 6 14% nd nd  5% nd  0% 15% nd  9% Spec 6 90%nd nd 90% nd 90% 90% nd 90% OR Quart 2 >3.3 nd nd 4.1 nd >13 >4.4nd >5.8 p Value <0.31 nd nd 0.094 nd <0.019 <0.20 nd <0.12 95% CIof >0.32 nd nd 0.79 nd >1.5 >0.46 nd >0.63 OR Quart 2 na nd nd 21 nd nana nd na OR Quart 3 >2.1 nd nd 8.3 nd >0 >4.4 nd >2.1 p Value <0.55 ndnd 0.0086 nd <na <0.20 nd <0.56 95% CI of >0.18 nd nd 1.7 nd >na >0.46nd >0.18 OR Quart 3 na nd nd 40 nd na na nd na OR Quart 4 >2.1 nd nd 0nd >19 >5.6 nd >4.4 p Value <0.56 nd nd na nd <0.0059 <0.12 nd <0.19 95%CI of >0.18 nd nd na nd >2.4 >0.62 nd >0.47 OR Quart 4 na nd nd na nd nana nd na C—X—C motif chemokine 6 24 hr prior to 48 hr prior to 0 hrprior to AKI stage AKI stage AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort2 Cohort 1 Cohort 2 sCr or UO Median 27.2 43.7 27.2 29.1 27.2 32.9Average 42.6 88.3 42.6 49.9 42.6 66.7 Stdev 42.3 86.5 42.3 55.1 42.383.1 p (t-test) 0.0100 0.48 0.080 Min 3.49 8.06 3.49 9.69 3.49 9.55 Max244 258 244 244 244 311 n (Samp) 131 7 131 21 131 13 n (Patient) 103 7103 21 103 13 UO only Median nd nd 25.5 29.1 25.5 24.0 Average nd nd40.5 44.7 40.5 61.1 Stdev nd nd 42.9 50.5 42.9 87.4 p (t-test) nd nd0.69 0.18 Min nd nd 3.49 8.06 3.49 9.69 Max nd nd 244 244 244 311 n(Samp) nd nd 113 21 113 11 n (Patient) nd nd 88 21 88 11 0 hr prior toAKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UOsCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO onlyAUC 0.68 nd nd 0.54 nd 0.55 0.59 nd 0.57 SE 0.11 nd nd 0.069 nd 0.0700.087 nd 0.094 p 0.11 nd nd 0.55 nd 0.46 0.28 nd 0.48 nCohort 1 131 ndnd 131 nd 113 131 nd 113 nCohort 2 7 nd nd 21 nd 21 13 nd 11 Cutoff 138.9 nd nd 17.6 nd 17.6 23.3 nd 23.3 Sens 1 71% nd nd 71% nd 71% 77% nd73% Spec 1 62% nd nd 34% nd 36% 44% nd 47% Cutoff 2 31.8 nd nd 15.8 nd15.8 17.6 nd 17.6 Sens 2 86% nd nd 81% nd 81% 85% nd 82% Spec 2 56% ndnd 25% nd 29% 34% nd 36% Cutoff 3 6.68 nd nd 13.9 nd 13.9 16.7 nd 16.7Sens 3 100%  nd nd 90% nd 90% 92% nd 91% Spec 3  5% nd nd 19% nd 24% 31%nd 34% Cutoff 4 46.0 nd nd 46.0 nd 43.7 46.0 nd 43.7 Sens 4 43% nd nd33% nd 33% 38% nd 27% Spec 4 70% nd nd 70% nd 72% 70% nd 72% Cutoff 560.9 nd nd 60.9 nd 54.2 60.9 nd 54.2 Sens 5 43% nd nd 29% nd 24% 31% nd27% Spec 5 80% nd nd 80% nd 81% 80% nd 81% Cutoff 6 93.3 nd nd 93.3 nd89.9 93.3 nd 89.9 Sens 6 43% nd nd 10% nd  5% 23% nd 18% Spec 6 90% ndnd 90% nd 90% 90% nd 90% OR Quart 2 0 nd nd 1.6 nd 2.6 5.6 nd 5.8 pValue na nd nd 0.50 nd 0.20 0.12 nd 0.12 95% CI of na nd nd 0.41 nd 0.610.62 nd 0.63 OR Quart 2 na nd nd 6.2 nd 11 51 nd 53 OR Quart 3 3.2 nd nd1.3 nd 1.8 3.2 nd 2.1 p Value 0.33 nd nd 0.72 nd 0.46 0.33 nd 0.56 95%CI of 0.32 nd nd 0.32 nd 0.39 0.31 nd 0.18 OR Quart 3 32 nd nd 5.2 nd8.2 32 nd 24 OR Quart 4 3.1 nd nd 1.6 nd 2.1 4.4 nd 3.2 p Value 0.34 ndnd 0.50 nd 0.31 0.20 nd 0.32 95% CI of 0.31 nd nd 0.41 nd 0.49 0.46 nd0.32 OR Quart 4 31 nd nd 6.2 nd 9.4 41 nd 33 Coagulation factor VII 24hr prior to 48 hr prior to 0 hr prior to AKI stage AKI stage AKI stageCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median310 281 310 235 310 221 Average 336 290 336 259 336 277 Stdev 172 149172 173 172 188 p (t-test) 0.19 0.014 0.17 Min 2.08 51.4 2.08 22.9 2.0812.2 Max 1020 549 1020 626 1020 616 n (Samp) 437 26 437 33 437 17 n(Patient) 174 26 174 33 174 17 sCr only Median 303 188 303 251 303 189Average 323 244 323 296 323 317 Stdev 174 189 174 235 174 219 p (t-test)0.27 0.65 0.93 Min 2.08 77.8 2.08 66.2 2.08 110 Max 1020 605 1020 7791020 622 n (Samp) 535 6 535 9 535 7 n (Patient) 207 6 207 9 207 7 UOonly Median 302 286 302 235 302 193 Average 321 310 321 253 321 247Stdev 163 155 163 168 163 172 p (t-test) 0.75 0.026 0.070 Min 16.0 51.416.0 22.9 16.0 12.2 Max 1020 611 1020 626 1020 616 n (Samp) 363 26 36331 363 17 n (Patient) 141 26 141 31 141 17 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.43 0.34 0.490.37 0.42 0.38 0.40 0.46 0.36 SE 0.060 0.12 0.059 0.054 0.10 0.056 0.0740.11 0.074 p 0.28 0.19 0.87 0.017 0.42 0.028 0.16 0.71 0.064 nCohort 1437 535 363 437 535 363 437 535 363 nCohort 2 26 6 26 33 9 31 17 7 17Cutoff 1 173 126 173 126 130 126 127 166 126 Sens 1 73% 83% 73% 73% 78%74% 71% 71% 76% Spec 1 17% 11% 18%  9% 12% 10%  9% 20% 10% Cutoff 2 156126 163 79.5 74.8 99.7 116 156 116 Sens 2 81% 83% 81% 82% 89% 81% 82%86% 82% Spec 2 14% 11% 16%  3%  5%  6%  6% 17%  7% Cutoff 3 76.2 76.2136 56.6 65.3 64.8 66.2 108 66.2 Sens 3 92% 100%  92% 91% 100%  90% 94%100%  94% Spec 3  3%  5% 10%  2%  4%  2%  2%  8%  2% Cutoff 4 405 403387 405 403 387 405 403 387 Sens 4 27% 17% 38% 21% 22% 23% 29% 29% 24%Spec 4 70% 70% 70% 70% 70% 70% 70% 70% 70% Cutoff 5 481 469 449 481 469449 481 469 449 Sens 5  8% 17% 27% 12% 22% 10% 12% 29% 18% Spec 5 80%80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6 573 564 536 573 564 536 573 564536 Sens 6  0% 17%  8%  6% 11% 10% 12% 29%  6% Spec 6 90% 90% 90% 90%90% 90% 90% 90% 90% OR Quart 2 0.56 0 0.48 1.7 0.50 1.0 0.75 0.50 0.49 pValue 0.36 na 0.25 0.39 0.57 0.99 0.71 0.57 0.42 95% CI of 0.16 na 0.140.53 0.044 0.31 0.16 0.045 0.087 OR Quart 2 2.0 na 1.7 5.2 5.5 3.2 3.45.6 2.7 OR Quart 3 0.70 2.0 0.61 1.0 1.5 0.82 0.49 0.50 0.49 p Value0.56 0.57 0.40 1.0 0.65 0.76 0.42 0.57 0.42 95% CI of 0.22 0.18 0.190.28 0.25 0.24 0.088 0.044 0.087 OR Quart 3 2.3 23 1.9 3.5 9.2 2.8 2.75.5 2.7 OR Quart 4 1.5 3.1 1.2 3.3 1.5 2.6 2.1 1.5 2.4 p Value 0.44 0.330.78 0.025 0.65 0.063 0.24 0.65 0.16 95% CI of 0.54 0.32 0.42 1.2 0.250.95 0.61 0.25 0.71 OR Quart 4 4.0 30 3.1 9.5 9.2 7.0 7.2 9.3 8.0Insulin-like growth factor-binding protein 7 24 hr prior to 48 hr priorto 0 hr prior to AKI stage AKI stage AKI stage Cohort 1 Cohort 2 Cohort1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 68.2 77.5 68.2 93.6 68.275.4 Average 73.4 93.2 73.4 112 73.4 84.5 Stdev 33.2 49.8 33.2 89.8 33.234.5 p (t-test) 0.13 7.1E−5 0.20 Min 18.6 37.5 18.6 43.7 18.6 50.1 Max250 189 250 437 250 176 n (Samp) 197 7 197 21 197 16 n (Patient) 131 7131 21 131 16 UO only Median nd nd 73.5 97.8 73.5 88.5 Average nd nd76.5 117 76.5 92.9 Stdev nd nd 33.2 92.8 33.2 38.7 p (t-test) nd nd1.7E−4 0.091 Min nd nd 27.8 43.7 27.8 54.2 Max nd nd 250 437 250 176 n(Samp) nd nd 173 18 173 13 n (Patient) nd nd 111 18 111 13 0 hr prior toAKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UOsCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO onlyAUC 0.63 nd nd 0.66 nd 0.65 0.61 nd 0.65 SE 0.12 nd nd 0.068 nd 0.0730.077 nd 0.085 p 0.28 nd nd 0.021 nd 0.036 0.14 nd 0.084 nCohort 1 197nd nd 197 nd 173 197 nd 173 nCohort 2 7 nd nd 21 nd 18 16 nd 13 Cutoff 174.2 nd nd 57.2 nd 57.2 58.1 nd 58.1 Sens 1 71% nd nd 71% nd 72% 75% nd77% Spec 1 56% nd nd 40% nd 36% 40% nd 36% Cutoff 2 57.2 nd nd 53.5 nd52.0 56.1 nd 56.1 Sens 2 86% nd nd 81% nd 83% 81% nd 85% Spec 2 40% ndnd 34% nd 22% 37% nd 33% Cutoff 3 36.7 nd nd 49.8 nd 49.5 53.9 nd 55.2Sens 3 100%  nd nd 90% nd 94% 94% nd 92% Spec 3  8% nd nd 24% nd 18% 34%nd 32% Cutoff 4 84.3 nd nd 84.3 nd 85.9 84.3 nd 85.9 Sens 4 43% nd nd52% nd 61% 44% nd 54% Spec 4 70% nd nd 70% nd 71% 70% nd 71% Cutoff 591.7 nd nd 91.7 nd 97.1 91.7 nd 97.1 Sens 5 43% nd nd 52% nd 50% 31% nd38% Spec 5 80% nd nd 80% nd 80% 80% nd 80% Cutoff 6 114 nd nd 114 nd 114114 nd 114 Sens 6 29% nd nd 33% nd 39% 19% nd 31% Spec 6 90% nd nd 90%nd 90% 90% nd 90% OR Quart 2 1.0 nd nd 1.7 nd 0.72 5.4 nd >5.5 p Value1.0 nd nd 0.48 nd 0.67 0.13 nd <0.13 95% CI of 0.061 nd nd 0.39 nd 0.150.61 nd >0.61 OR Quart 2 16 nd nd 7.5 nd 3.4 48 nd na OR Quart 3 2.0 ndnd 0.65 nd 0 4.2 nd >2.1 p Value 0.57 nd nd 0.65 nd na 0.20 nd <0.55 95%CI of 0.18 nd nd 0.10 nd na 0.46 nd >0.18 OR Quart 3 23 nd nd 4.1 nd na39 nd na OR Quart 4 3.1 nd nd 4.2 nd 3.2 6.5 nd >6.7 p Value 0.33 nd nd0.034 nd 0.063 0.088 nd <0.083 95% CI of 0.31 nd nd 1.1 nd 0.94 0.75nd >0.78 OR Quart 4 31 nd nd 16 nd 11 56 nd na

TABLE 7 Comparison of marker levels in EDTA samples collected within 12hours of reaching stage R from Cohort 1 (patients that reached, but didnot progress beyond, RIFLE stage R) and from Cohort 2 (patients thatreached RIFLE stage I or F). Coagulation factor VII sCr or UO sCr onlyCohort Cohort Cohort Cohort UO only 1 2 1 2 Cohort 1 Cohort 2 Median 313250 240 333 324 279 Average 335 258 333 282 325 268 Stdev 191 169 213142 182 169 p (t-test) 0.10 0.57 0.28 Min 35.7 22.9 65.3 75.6 35.7 22.9Max 809 616 759 453 809 616 n (Samp) 55 22 18 7 46 16 n (Patient) 55 2218 7 46 16 At Enrollment sCr or UO sCr only UO only AUC 0.38 0.46 0.41SE 0.073 0.13 0.085 p 0.100 0.76 0.28 nCohort 1 55 18 46 nCohort 2 22 716 Cutoff 1 137 245 137 Sens 1 73% 71% 81% Spec 1 15% 56% 17% Cutoff 295.4 75.6 137 Sens 2 82% 86% 81% Spec 2  5%  6% 17% Cutoff 3 51.2 65.335.7 Sens 3 91% 100%  94% Spec 3  2%  6%  2% Cutoff 4 446 415 446 Sens 414% 14% 12% Spec 4 71% 72% 72% Cutoff 5 484 536 483 Sens 5 14%  0% 12%Spec 5 80% 83% 80% Cutoff 6 598 737 536 Sens 6  5%  0% 12% Spec 6 91%94% 91% OR Quart 2 3.3 12 3.5 p Value 0.13 0.073 0.18 95% CI of 0.710.80 0.56 OR Quart 2 15 180 22 OR Quart 3 1.5 0 2.3 p Value 0.62 na 0.3795% CI of 0.29 na 0.36 OR Quart 3 7.9 na 15 OR Quart 4 4.1 3.0 3.5 pValue 0.069 0.43 0.18 95% CI of 0.89 0.20 0.56 OR Quart 4 19 45 22

TABLE 8 Comparison of the maximum marker levels in EDTA samplescollected from Cohort 1 (patients that did not progress beyond RIFLEstage 0) and the maximum values in EDTA samples collected from subjectsbetween enrollment and 0, 24 hours, and 48 hours prior to reaching stageF in Cohort 2. Cancer Antigen 19-9 0 hr prior to AKI stage 24 hr priorto AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort2 Cohort 1 Cohort 2 sCr or UO Median 0.204 0.652 0.204 0.601 nd ndAverage 0.339 1.21 0.339 1.16 nd nd Stdev 0.668 1.66 0.668 1.67 nd nd p(t-test) 0.0046 0.0077 nd nd Min 1.00E−9 0.145 1.00E−9 0.145 nd nd Max5.30 5.26 5.30 5.26 nd nd n (Samp) 74 8 74 8 nd nd n (Patient) 74 8 74 8nd nd UO only Median 0.258 0.588 0.258 0.588 nd nd Average 0.472 1.330.472 1.33 nd nd Stdev 0.791 1.95 0.791 1.95 nd nd p (t-test) 0.0340.034 nd nd Min 1.00E−9 0.145 1.00E−9 0.145 nd nd Max 5.30 5.26 5.305.26 nd nd n (Samp) 64 6 64 6 nd nd n (Patient) 64 6 64 6 nd nd 0 hrprior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCror UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UOonly AUC 0.85 nd 0.74 0.84 nd 0.74 nd nd nd SE 0.088 nd 0.12 0.090 nd0.12 nd nd nd p 7.4E−5 nd 0.045 2.0E−4 nd 0.045 nd nd nd nCohort 1 74 nd64 74 nd 64 nd nd nd nCohort 2 8 nd 6 8 nd 6 nd nd nd Cutoff 1 0.546 nd0.358 0.546 nd 0.358 nd nd nd Sens 1 75% nd 83% 75% nd 83% nd nd nd Spec1 88% nd 69% 88% nd 69% nd nd nd Cutoff 2 0.358 nd 0.358 0.358 nd 0.358nd nd nd Sens 2 88% nd 83% 88% nd 83% nd nd nd Spec 2 80% nd 69% 80% nd69% nd nd nd Cutoff 3 0.124 nd 0.134 0.124 nd 0.134 nd nd nd Sens 3100%  nd 100%  100%  nd 100%  nd nd nd Spec 3 34% nd 28% 34% nd 28% ndnd nd Cutoff 4 0.311 nd 0.401 0.311 nd 0.401 nd nd nd Sens 4 88% nd 67%88% nd 67% nd nd nd Spec 4 70% nd 70% 70% nd 70% nd nd nd Cutoff 5 0.401nd 0.629 0.401 nd 0.629 nd nd nd Sens 5 75% nd 33% 75% nd 33% nd nd ndSpec 5 81% nd 81% 81% nd 81% nd nd nd Cutoff 6 0.629 nd 0.944 0.629 nd0.944 nd nd nd Sens 6 50% nd 33% 38% nd 33% nd nd nd Spec 6 91% nd 91%91% nd 91% nd nd nd OR Quart 2 >1.0 nd >1.0 >1.0 nd >1.0 nd nd nd pValue <1.0 nd <1.0 <1.0 nd <1.0 nd nd nd 95% CI of >0.058nd >0.058 >0.058 nd >0.058 nd nd nd OR Quart 2 na nd na na nd na nd ndnd OR Quart 3 >1.1 nd >1.1 >1.1 nd >1.1 nd nd nd p Value <0.97 nd <0.97<0.97 nd <0.97 nd nd nd 95% CI of >0.061 nd >0.061 >0.061 nd >0.061 ndnd nd OR Quart 3 na nd na na nd na nd nd nd OR Quart 4 >8.0 nd >4.9 >8.0nd >4.9 nd nd nd p Value <0.066 nd <0.18 <0.066 nd <0.18 nd nd nd 95% CIof >0.87 nd >0.49 >0.87 nd >0.49 nd nd nd OR Quart 4 na nd na na nd nand nd nd C-C motif chemokine 13 0 hr prior to AKI stage 24 hr prior toAKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 21.0 42.6 21.0 42.6 21.0 42.6 Average104 90.3 104 90.3 104 42.9 Stdev 371 199 371 199 371 32.6 p (t-test)0.90 0.90 0.69 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max2520 713 2520 713 2520 96.3 n (Samp) 50 12 50 12 50 6 n (Patient) 50 1250 12 50 6 sCr only Median 21.0 1.00E−9 21.0 1.00E−9 nd nd Average 72.520.5 72.5 20.5 nd nd Stdev 268 32.0 268 32.0 nd nd p (t-test) 0.64 0.64nd nd Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 nd nd Max 2520 68.2 2520 68.2nd nd n (Samp) 100 6 100 6 nd nd n (Patient) 100 6 100 6 nd nd UO onlyMedian 21.0 44.5 21.0 44.5 21.0 42.6 Average 115 127 115 127 115 42.9Stdev 387 239 387 239 387 32.6 p (t-test) 0.93 0.93 0.65 Min 1.00E−91.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 2520 713 2520 713 2520 96.3n (Samp) 46 8 46 8 46 6 n (Patient) 46 8 46 8 46 6 0 hr prior to AKIstage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UO sCronly UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC0.54 0.40 0.63 0.54 0.40 0.63 0.58 nd 0.58 SE 0.095 0.13 0.11 0.095 0.130.11 0.13 nd 0.13 p 0.69 0.41 0.25 0.69 0.41 0.25 0.53 nd 0.56 nCohort 150 100 46 50 100 46 50 nd 46 nCohort 2 12 6 8 12 6 8 6 nd 6 Cutoff 1 0 038.0 0 0 38.0 7.19 nd 7.19 Sens 1 100%  100%  75% 100%  100%  75% 83% nd83% Spec 1  0%  0% 61%  0%  0% 61% 44% nd 43% Cutoff 2 0 0 7.19 0 0 7.197.19 nd 7.19 Sens 2 100%  100%  88% 100%  100%  88% 83% nd 83% Spec 2 0%  0% 43%  0%  0% 43% 44% nd 43% Cutoff 3 0 0 0 0 0 0 0 nd 0 Sens 3100%  100%  100%  100%  100%  100%  100%  nd 100%  Spec 3  0%  0%  0% 0%  0%  0%  0% nd  0% Cutoff 4 56.0 44.5 59.5 56.0 44.5 59.5 56.0 nd59.5 Sens 4 25% 33% 25% 25% 33% 25% 17% nd 17% Spec 4 70% 70% 72% 70%70% 72% 70% nd 72% Cutoff 5 72.2 69.2 69.2 72.2 69.2 69.2 72.2 nd 69.2Sens 5 17%  0% 25% 17%  0% 25% 17% nd 17% Spec 5 80% 81% 80% 80% 81% 80%80% nd 80% Cutoff 6 105 96.3 105 105 96.3 105 105 nd 105 Sens 6  8%  0%12%  8%  0% 12%  0% nd  0% Spec 6 90% 90% 91% 90% 90% 91% 90% nd 91% ORQuart 2 0.18 1.0 0.92 0.18 1.0 0.92 1.0 nd 1.0 p Value 0.15 0.98 0.960.15 0.98 0.96 1.0 nd 1.0 95% CI of 0.018 0.062 0.052 0.018 0.062 0.0520.056 nd 0.056 OR Quart 2 1.9 18 16 1.9 18 16 18 nd 18 OR Quart 3 1.04.5 5.3 1.0 4.5 5.3 3.5 nd 3.6 p Value 1.0 0.19 0.16 1.0 0.19 0.16 0.30nd 0.30 95% CI of 0.20 0.47 0.51 0.20 0.47 0.51 0.32 nd 0.32 OR Quart 35.0 43 56 5.0 43 56 39 nd 40 OR Quart 4 0.63 0 2.0 0.63 0 2.0 1.0 nd 1.0p Value 0.60 na 0.59 0.60 na 0.59 1.0 nd 1.0 95% CI of 0.12 na 0.16 0.12na 0.16 0.056 nd 0.056 OR Quart 4 3.5 na 25 3.5 na 25 18 nd 18 C—X—Cmotif chemokine 6 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hrprior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2sCr or UO Median 34.7 43.2 34.7 43.2 34.7 46.7 Average 49.8 64.5 49.864.5 49.8 45.7 Stdev 49.7 87.6 49.7 87.6 49.7 21.8 p (t-test) 0.44 0.440.84 Min 5.55 9.69 5.55 9.69 5.55 17.9 Max 244 336 244 336 244 72.4 n(Samp) 51 12 51 12 51 6 n (Patient) 51 12 51 12 51 6 sCr only Median27.2 33.4 27.2 33.4 nd nd Average 45.3 34.9 45.3 34.9 nd nd Stdev 50.522.4 50.5 22.4 nd nd p (t-test) 0.62 0.62 nd nd Min 4.02 9.69 4.02 9.69nd nd Max 311 72.4 311 72.4 nd nd n (Samp) 101 6 101 6 nd nd n (Patient)101 6 101 6 nd nd UO only Median 31.0 46.7 31.0 46.7 31.0 46.7 Average45.7 81.8 45.7 81.8 45.7 45.7 Stdev 49.1 105 49.1 105 49.1 21.8 p(t-test) 0.12 0.12 1.00 Min 5.55 17.9 5.55 17.9 5.55 17.9 Max 244 336244 336 244 72.4 n (Samp) 47 8 47 8 47 6 n (Patient) 47 8 47 8 47 6 0 hrprior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage sCror UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UOonly AUC 0.59 0.51 0.70 0.59 0.51 0.70 0.60 nd 0.65 SE 0.095 0.12 0.110.095 0.12 0.11 0.13 nd 0.13 p 0.36 0.92 0.063 0.36 0.92 0.063 0.42 nd0.24 nCohort 1 51 101 47 51 101 47 51 nd 47 nCohort 2 12 6 8 12 6 8 6 nd6 Cutoff 1 25.5 17.6 42.8 25.5 17.6 42.8 24.0 nd 24.0 Sens 1 75% 83% 75%75% 83% 75% 83% nd 83% Spec 1 39% 32% 70% 39% 32% 70% 33% nd 38% Cutoff2 24.1 17.6 24.1 24.1 17.6 24.1 24.0 nd 24.0 Sens 2 83% 83% 88% 83% 83%88% 83% nd 83% Spec 2 37% 32% 40% 37% 32% 40% 33% nd 38% Cutoff 3 17.68.88 17.6 17.6 8.88 17.6 17.6 nd 17.6 Sens 3 92% 100%  100%  92% 100% 100%  100%  nd 100%  Spec 3 27% 10% 30% 27% 10% 30% 27% nd 30% Cutoff 446.9 43.7 42.8 46.9 43.7 42.8 46.9 nd 42.8 Sens 4 33% 17% 75% 33% 17%75% 50% nd 67% Spec 4 71% 70% 70% 71% 70% 70% 71% nd 70% Cutoff 5 56.756.7 51.1 56.7 56.7 51.1 56.7 nd 51.1 Sens 5 25% 17% 38% 25% 17% 38% 33%nd 33% Spec 5 80% 80% 81% 80% 80% 81% 80% nd 81% Cutoff 6 117 95.2 117117 95.2 117 117 nd 117 Sens 6  8%  0% 12%  8%  0% 12%  0% nd  0% Spec 690% 90% 91% 90% 90% 91% 90% nd 91% OR Quart 2 3.2 2.0 >2.2 3.22.0 >2.2 >2.3 nd >2.4 p Value 0.34 0.58 <0.55 0.34 0.58 <0.55 <0.51 nd<0.51 95% CI of 0.30 0.17 >0.17 0.30 0.17 >0.17 >0.19 nd >0.19 OR Quart2 35 23 na 35 23 na na nd na OR Quart 3 6.4 2.0 >2.2 6.4 2.0 >2.2 >2.3nd >1.1 p Value 0.11 0.58 <0.55 0.11 0.58 <0.55 <0.51 nd <0.96 95% CI of0.65 0.17 >0.17 0.65 0.17 >0.17 >0.19 nd >0.061 OR Quart 3 63 23 na 6323 na na nd na OR Quart 4 3.2 0.96 >5.2 3.2 0.96 >5.2 >2.2 nd >3.5 pValue 0.34 0.98 <0.17 0.34 0.98 <0.17 <0.55 nd <0.30 95% CI of 0.300.057 >0.50 0.30 0.057 >0.50 >0.17 nd >0.32 OR Quart 4 35 16 na 35 16 nana nd na Coagulation factor VII 0 hr prior to AKI stage 24 hr prior toAKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 349 309 349 246 349 230 Average 382318 382 274 382 298 Stdev 165 193 165 179 165 165 p (t-test) 0.16 0.0160.17 Min 2.33 32.8 2.33 32.8 2.33 139 Max 743 779 743 779 743 622 n(Samp) 110 16 110 16 110 8 n (Patient) 110 16 110 16 110 8 sCr onlyMedian 344 244 344 222 nd nd Average 368 261 368 231 nd nd Stdev 170 118170 103 nd nd p (t-test) 0.079 0.025 nd nd Min 2.33 77.8 2.33 75.6 nd ndMax 822 430 822 386 nd nd n (Samp) 180 8 180 8 nd nd n (Patient) 180 8180 8 nd nd UO only Median 356 358 356 310 356 304 Average 388 368 388321 388 324 Stdev 169 215 169 206 169 186 p (t-test) 0.73 0.25 0.38 Min119 32.8 119 32.8 119 139 Max 1020 779 1020 779 1020 622 n (Samp) 91 1091 10 91 6 n (Patient) 91 10 91 10 91 6 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.39 0.32 0.480.31 0.25 0.39 0.34 nd 0.40 SE 0.079 0.11 0.098 0.077 0.10 0.099 0.11 nd0.13 p 0.17 0.086 0.82 0.011 0.015 0.28 0.14 nd 0.43 nCohort 1 110 18091 110 180 91 110 nd 91 nCohort 2 16 8 10 16 8 10 8 nd 6 Cutoff 1 185190 283 151 190 246 195 nd 150 Sens 1 75% 75% 70% 75% 75% 70% 75% nd 83%Spec 1 11% 15% 26%  9% 15% 19% 13% nd  7% Cutoff 2 173 177 274 138 126150 151 nd 150 Sens 2 81% 88% 80% 81% 88% 80% 88% nd 83% Spec 2 10% 14%24%  5%  4%  7%  9% nd  7% Cutoff 3 32.8 53.5 138 32.8 53.5 138 138 nd138 Sens 3 94% 100%  90% 94% 100%  90% 100%  nd 100%  Spec 3  1%  2%  4% 1%  2%  4%  5% nd  4% Cutoff 4 456 456 437 456 456 437 456 nd 437 Sens4 12%  0% 30%  6%  0% 20% 12% nd 17% Spec 4 70% 71% 70% 70% 71% 70% 70%nd 70% Cutoff 5 533 510 549 533 510 549 533 nd 549 Sens 5 12%  0% 20% 6%  0% 10% 12% nd 17% Spec 5 80% 80% 80% 80% 80% 80% 80% nd 80% Cutoff6 646 623 632 646 623 632 646 nd 632 Sens 6  6%  0% 10%  6%  0% 10%  0%nd  0% Spec 6 90% 90% 90% 90% 90% 90% 90% nd 90% OR Quart 2 2.2 >2.1 1.63.3 >1.0 3.4 2.1 nd 2.2 p Value 0.38 <0.55 0.61 0.31 <0.99 0.30 0.54 nd0.54 95% CI of 0.38 >0.18 0.25 0.33 >0.062 0.33 0.18 nd 0.18 OR Quart 213 na 11 34 na 35 25 nd 26 OR Quart 3 2.1 >2.1 1.6 4.4 >3.2 2.2 0 nd 0 pValue 0.40 <0.55 0.61 0.19 <0.32 0.54 na nd na 95% CI of 0.36 >0.18 0.250.47 >0.32 0.18 na nd na OR Quart 3 13 na 11 42 na 26 na nd na OR Quart4 3.6 >4.4 1.0 11 >4.4 4.8 6.0 nd 3.4 p Value 0.14 <0.19 0.97 0.030<0.19 0.18 0.11 nd 0.30 95% CI of 0.67 >0.47 0.14 1.3 >0.47 0.49 0.66 nd0.33 OR Quart 4 19 na 8.0 92 na 46 55 nd 36 Insulin-like growthfactor-binding protein 7 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 65.8 160 65.8 132 nd nd Average 70.2181 70.2 174 nd nd Stdev 35.5 120 35.5 121 nd nd p (t-test) 4.1E−82.2E−7 nd nd Min 18.6 58.3 18.6 58.3 nd nd Max 250 437 250 437 nd nd n(Samp) 74 8 74 8 nd nd n (Patient) 74 8 74 8 nd nd UO only Median 73.5114 73.5 114 nd nd Average 76.8 171 76.8 171 nd nd Stdev 35.4 140 35.4140 nd nd p (t-test) 5.0E−5 5.0E−5 nd nd Min 28.5 58.3 28.5 58.3 nd ndMax 250 437 250 437 nd nd n (Samp) 64 6 64 6 nd nd n (Patient) 64 6 64 6nd nd 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior toAKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr orUO sCr only UO only AUC 0.89 nd 0.82 0.88 nd 0.82 nd nd nd SE 0.079 nd0.11 0.080 nd 0.11 nd nd nd p 9.7E−7 nd 0.0029 1.6E−6 nd 0.0029 nd nd ndnCohort 1 74 nd 64 74 nd 64 nd nd nd nCohort 2 8 nd 6 8 nd 6 nd nd ndCutoff 1 96.9 nd 91.7 96.9 nd 91.7 nd nd nd Sens 1 75% nd 83% 75% nd 83%nd nd nd Spec 1 86% nd 81% 86% nd 81% nd nd nd Cutoff 2 91.7 nd 91.791.7 nd 91.7 nd nd nd Sens 2 88% nd 83% 88% nd 83% nd nd nd Spec 2 85%nd 81% 85% nd 81% nd nd nd Cutoff 3 57.2 nd 58.1 57.2 nd 58.1 nd nd ndSens 3 100%  nd 100%  100%  nd 100%  nd nd nd Spec 3 45% nd 36% 45% nd36% nd nd nd Cutoff 4 79.2 nd 84.3 79.2 nd 84.3 nd nd nd Sens 4 88% nd83% 88% nd 83% nd nd nd Spec 4 70% nd 70% 70% nd 70% nd nd nd Cutoff 587.0 nd 91.7 87.0 nd 91.7 nd nd nd Sens 5 88% nd 83% 88% nd 83% nd nd ndSpec 5 81% nd 81% 81% nd 81% nd nd nd Cutoff 6 114 nd 115 114 nd 115 ndnd nd Sens 6 62% nd 50% 62% nd 50% nd nd nd Spec 6 91% nd 91% 91% nd 91%nd nd nd OR Quart 2 >1.0 nd >1.0 >1.0 nd >1.0 nd nd nd p Value <1.0 nd<1.0 <1.0 nd <1.0 nd nd nd 95% CI of >0.058 nd >0.058 >0.058 nd >0.058nd nd nd OR Quart 2 na nd na na nd na nd nd nd OR Quart 3 >0 nd >0 >0nd >0 nd nd nd p Value <na nd <na <na nd <na nd nd nd 95% CI of >nand >na >na nd >na nd nd nd OR Quart 3 na nd na na nd na nd nd nd ORQuart 4 >10 nd >6.5 >10 nd >6.5 nd nd nd p Value <0.041 nd <0.10 <0.041nd <0.10 nd nd nd 95% CI of >1.1 nd >0.68 >1.1 nd >0.68 nd nd nd ORQuart 4 na nd na na nd na nd nd nd

TABLE 9 Comparison of marker levels in urine samples collected fromCohort 1 (patients that did not progress beyond RIFLE stage 0, R, or I)and in urine samples collected from Cohort 2 (subjects who progress toRIFLE stage F) at 0, 24 hours, and 48 hours prior to the subjectreaching RIFLE stage I. Cancer Antigen 19-9 0 hr prior to AKI stage 24hr prior to AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 22.9 38.8 22.9 42.2 nd ndAverage 145 35.3 145 391 nd nd Stdev 710 21.1 710 917 nd nd p(t-test)0.68 0.28 nd nd Min 1.00E−9 6.51 1.00E−9 18.6 nd nd Max 11900 59.3 119002940 nd nd n (Samp) 324 7 324 10 nd nd n (Patient) 190 7 190 10 nd nd UOonly Median nd nd 25.8 38.7 nd nd Average nd nd 156 476 nd nd Stdev ndnd 755 1020 nd nd p(t-test) nd nd 0.24 nd nd Min nd nd 1.00E−9 18.6 ndnd Max nd nd 11900 2940 nd nd n (Samp) nd nd 283 8 nd nd n (Patient) ndnd 159 8 nd nd 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hrprior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO onlysCr or UO sCr only UO only AUC 0.54 nd nd 0.67 nd 0.65 nd nd nd SE 0.11nd nd 0.095 nd 0.11 nd nd nd p 0.71 nd nd 0.068 nd 0.16 nd nd nd nCohort1 324 nd nd 324 nd 283 nd nd nd nCohort 2 7 nd nd 10 nd 8 nd nd ndCutoff 1 36.7 nd nd 27.5 nd 21.8 nd nd nd Sens 1 71% nd nd 70% nd 75% ndnd nd Spec 1 64% nd nd 56% nd 45% nd nd nd Cutoff 2 6.59 nd nd 21.9 nd20.7 nd nd nd Sens 2 86% nd nd 80% nd 88% nd nd nd Spec 2 22% nd nd 48%nd 43% nd nd nd Cutoff 3 6.42 nd nd 20.7 nd 18.5 nd nd nd Sens 3 100% nd nd 90% nd 100%  nd nd nd Spec 3 21% nd nd 47% nd 41% nd nd nd Cutoff4 54.2 nd nd 54.2 nd 57.7 nd nd nd Sens 4 14% nd nd 40% nd 38% nd nd ndSpec 4 70% nd nd 70% nd 70% nd nd nd Cutoff 5 102 nd nd 102 nd 116 nd ndnd Sens 5  0% nd nd 20% nd 25% nd nd nd Spec 5 80% nd nd 80% nd 80% ndnd nd Cutoff 6 240 nd nd 240 nd 277 nd nd nd Sens 6  0% nd nd 20% nd 25%nd nd nd Spec 6 90% nd nd 90% nd 90% nd nd nd OR Quart 2 0 nd nd >3.1nd >3.1 nd nd nd p Value na nd nd <0.34 nd <0.33 nd nd nd 95% CI of nand nd >0.31 nd >0.31 nd nd nd OR Quart2 na nd nd na nd na nd nd nd ORQuart 3 2.6 nd nd >3.1 nd >2.0 nd nd nd p Value 0.27 nd nd <0.33 nd<0.57 nd nd nd 95% CI of 0.48 nd nd >0.32 nd >0.18 nd nd nd OR Quart3 14nd nd na nd na nd nd nd OR Quart 4 0 nd nd >4.2 nd >3.1 nd nd nd p Valuena nd nd <0.21 nd <0.33 nd nd nd 95% CI of na nd nd >0.45 nd >0.31 nd ndnd OR Quart4 na nd nd na nd na nd nd nd C-C motif chemokine 13 0 hrprior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKI stageCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median1.00E−9 1.00E−9 1.00E−9 3.52 1.00E−9 1.00E−9 Average 2.67 13.6 2.67 22.22.67 8.85 Stdev 16.4 40.1 16.4 35.5 16.4 23.4 p(t-test) 0.021 5.2E−6 0.32 Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 1.00E−9 Max 241 143 241101 241 61.9 n (Samp) 937 13 937 16 937 7 n (Patient) 345 13 345 16 3457 sCr only Median 1.00E−9 1.00E−9 nd nd nd nd Average 3.18 1.00E−9 nd ndnd nd Stdev 17.6 0 nd nd nd nd p(t-test) 0.63 nd nd nd nd Min 1.00E−91.00E−9 nd nd nd nd Max 241 1.00E−9 nd nd nd nd n (Samp) 974 7 nd nd ndnd n (Patient) 355 7 nd nd nd nd UO only Median 1.00E−9 1.00E−9 1.00E−93.52 nd nd Average 2.68 17.9 2.68 24.8 nd nd Stdev 17.0 50.7 17.0 37.3nd nd p(t-test) 0.015 3.1E−6  nd nd Min 1.00E−9 1.00E−9 1.00E−9 1.00E−9nd nd Max 241 143 241 101 nd nd n (Samp) 804 8 804 14 nd nd n (Patient)264 8 264 14 nd nd 0 hr prior to AKI stage 24 hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UOonly sCr or UO sCr only UO only AUC 0.55 0.46 0.53 0.72 nd 0.72 0.54 ndnd SE 0.083 0.11 0.10 0.073 nd 0.078 0.11 nd nd p 0.56 0.75 0.74 0.0028nd 0.0046 0.71 nd nd nCohort 1 937 974 804 937 nd 804 937 nd nd nCohort2 13 7 8 16 nd 14 7 nd nd Cutoff 1 0 0 0 0 nd 0 0 nd nd Sens 1 100% 100%  100%  100%  nd 100%  100%  nd nd Spec 1  0% 0%  0%  0% nd  0%  0%nd nd Cutoff 2 0 0 0 0 nd 0 0 nd nd Sens 2 100%  100%  100%  100%  nd100%  100%  nd nd Spec 2  0% 0%  0%  0% nd  0%  0% nd nd Cutoff 3 0 0 00 nd 0 0 nd nd Sens 3 100%  100%  100%  100%  nd 100%  100%  nd nd Spec3  0% 0%  0%  0% nd  0%  0% nd nd Cutoff 4 1.00E−9 1.00E−9 1.00E−91.00E−9 nd 1.00E−9 1.00E−9 nd nd Sens 4 15% 0% 12% 50% nd 50% 14% nd ndSpec 4 93% 93%  94% 93% nd 94% 93% nd nd Cutoff 5 1.00E−9 1.00E−91.00E−9 1.00E−9 nd 1.00E−9 1.00E−9 nd nd Sens 5 15% 0% 12% 50% nd 50%14% nd nd Spec 5 93% 93%  94% 93% nd 94% 93% nd nd Cutoff 6 1.00E−91.00E−9 1.00E−9 1.00E−9 nd 1.00E−9 1.00E−9 nd nd Sens 6 15% 0% 12% 50%nd 50% 14% nd nd Spec 6 93% 93%  94% 93% nd 94% 93% nd nd OR Quart2 >11 >0 >7.2 >8.3 nd >7.2 >6.2 nd nd p Value <0.020 <na <0.065 <0.047nd <0.066 <0.094 nd nd 95% CI of >1.5 >na >0.88 >1.0 nd >0.88 >0.74 ndnd OR Quart2 na   na na na nd na na nd nd OR Quart 3 >0 >7.2 >0 >0nd >0 >0 nd nd p Value <na   <0.065 <na   <na   nd <na   <na   nd nd 95%CI of >na   >0.88 >na   >na   nd >na   >na   nd nd OR Quart3 na   na nana nd na na nd nd OR Quart 4 >2.0 >0 >1.0 >8.2 nd >7.2 >1.0 nd nd pValue <0.57 <na <1.00 <0.048 nd <0.066 <1.00 nd nd 95% CIof >0.18 >na >0.062 >1.0 nd >0.88 >0.062 nd nd OR Quart4 na   na na nand na na nd nd C-X-C motif chemokine 6 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 15.5 19.3 15.5 82.0 15.58.72 Average 35.9 206 35.9 201 35.9 26.6 Stdev 106 453 106 252 106 50.3p(t-test) 2.4E−7 3.5E−9 0.82 Min 1.00E−9 1.62 1.00E−9 9.04 1.00E−9 0.506Max 2500 1450 2500 769 2500 140 n (Samp) 935 13 935 16 935 7 n (Patient)345 13 345 16 345 7 sCr only Median 15.9 61.3 nd nd nd nd Average 40.3281 nd nd nd nd Stdev 116 530 nd nd nd nd p(t-test) 2.8E−7 nd nd nd ndMin 1.00E−9 1.62 nd nd nd nd Max 2500 1450 nd nd nd nd n (Samp) 972 7 ndnd nd nd n (Patient) 355 7 nd nd nd nd UO only Median 17.0 40.1 17.099.7 nd nd Average 37.5 320 37.5 225 nd nd Stdev 112 560 112 262 nd ndp(t-test)  2.0E−10 3.5E−9 nd nd Min 1.00E−9 3.96 1.00E−9 9.04 nd nd Max2500 1450 2500 769 nd nd n (Samp) 802 8 802 14 nd nd n (Patient) 264 8264 14 nd nd 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hrprior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO onlysCr or UO sCr only UO only AUC 0.58 0.65 0.62 0.83 nd 0.84 0.37 nd nd SE0.083 0.11 0.11 0.063 nd 0.067 0.11 nd nd p 0.36 0.20 0.28 2.1E−7 nd4.1E−7 0.25 nd nd nCohort 1 935 972 802 935 nd 802 935 nd nd nCohort 213 7 8 16 nd 14 7 nd nd Cutoff 1 7.87 11.6 7.87 48.3 nd 51.2 4.01 nd ndSens 1 77% 71% 75% 75% nd 71% 71% nd nd Spec 1 31% 39% 28% 84% nd 85%17% nd nd Cutoff 2 5.34 7.91 5.34 24.7 nd 24.5 0.534 nd nd Sens 2 85%86% 88% 81% nd 86% 86% nd nd Spec 2 22% 31% 19% 64% nd 62%  5% nd ndCutoff 3 3.84 1.62 3.83 12.0 nd 12.0 0.437 nd nd Sens 3 92% 100%  100% 94% nd 93% 100%  nd nd Spec 3 16%  8% 13% 41% nd 38%  4% nd nd Cutoff 429.2 30.3 30.1 29.2 nd 30.1 29.2 nd nd Sens 4 46% 57% 50% 75% nd 79% 14%nd nd Spec 4 70% 70% 70% 70% nd 70% 70% nd nd Cutoff 5 40.3 43.6 41.140.3 nd 41.1 40.3 nd nd Sens 5 38% 57% 50% 75% nd 79% 14% nd nd Spec 580% 80% 80% 80% nd 80% 80% nd nd Cutoff 6 63.9 69.4 63.9 63.9 nd 63.963.9 nd nd Sens 6 23% 29% 25% 56% nd 64% 14% nd nd Spec 6 90% 90% 90%90% nd 90% 90% nd nd OR Quart 2 1.0 2.0 0.50 >2.0 nd >2.0 1.0 nd nd pValue 1.0 0.57 0.57 <0.57 nd <0.57 1.00 nd nd 95% CI of 0.20 0.180.045 >0.18 nd >0.18 0.062 nd nd OR Quart2 5.0 22 5.5 na nd na 16 nd ndOR Quart 3 0.66 0 0.50 >2.0 nd >1.0 2.0 nd nd p Value 0.66 na 0.57 <0.57nd <1.00 0.57 nd nd 95% CI of 0.11 na 0.045 >0.18 nd >0.062 0.18 nd ndOR Quart3 4.0 na 5.5 na nd na 22 nd nd OR Quart 4 1.7 4.0 2.0 >13 nd >123.0 nd nd p Value 0.48 0.21 0.42 <0.015 nd <0.019 0.34 nd nd 95% CI of0.40 0.45 0.36 >1.6 nd >1.5 0.31 nd nd OR Quart4 7.1 36 11 na nd na 29nd nd Coagulation factor VII 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 3.14 3.73 3.14 2.84 nd nd Average6.63 5.14 6.63 7.07 nd nd Stdev 13.6 5.61 13.6 8.20 nd nd p(t-test) 0.730.92 nd nd Min 0.00408 0.348 0.00408 0.682 nd nd Max 249 18.9 249 24.9nd nd n (Samp) 534 10 534 11 nd nd n (Patient) 204 10 204 11 nd nd UOonly Median 3.08 4.33 3.08 2.79 nd nd Average 6.47 4.83 6.47 5.67 nd ndStdev 14.1 2.60 14.1 6.68 nd nd p(t-test) 0.76 0.87 nd nd Min 0.004080.550 0.00408 0.682 nd nd Max 249 8.18 249 19.4 nd nd n (Samp) 454 7 4548 nd nd n (Patient) 168 7 168 8 nd nd 0 hr prior to AKI stage 24 hrprior to AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO onlysCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.48 nd 0.570.55 nd 0.52 nd nd nd SE 0.093 nd 0.11 0.090 nd 0.10 nd nd nd p 0.87 nd0.52 0.55 nd 0.88 nd nd nd nCohort 1 534 nd 454 534 nd 454 nd nd ndnCohort 2 10 nd 7 11 nd 8 nd nd nd Cutoff 1 2.27 nd 4.15 2.34 nd 2.34 ndnd nd Sens 1 70% nd 71% 73% nd 75% nd nd nd Spec 1 40% nd 59% 41% nd 42%nd nd nd Cutoff 2 0.539 nd 3.28 2.16 nd 1.50 nd nd nd Sens 2 80% nd 86%82% nd 88% nd nd nd Spec 2 11% nd 51% 39% nd 31% nd nd nd Cutoff 3 0.420nd 0.539 1.50 nd 0.676 nd nd nd Sens 3 90% nd 100%  91% nd 100%  nd ndnd Spec 3  7% nd 11% 31% nd 13% nd nd nd Cutoff 4 6.36 nd 6.11 6.36 nd6.11 nd nd nd Sens 4 30% nd 29% 27% nd 25% nd nd nd Spec 4 70% nd 70%70% nd 70% nd nd nd Cutoff 5 9.31 nd 8.93 9.31 nd 8.93 nd nd nd Sens 510% nd  0% 27% nd 25% nd nd nd Spec 5 80% nd 80% 80% nd 80% nd nd ndCutoff 6 15.0 nd 14.3 15.0 nd 14.3 nd nd nd Sens 6 10% nd  0% 18% nd 12%nd nd nd Spec 6 90% nd 91% 90% nd 91% nd nd nd OR Quart 2 2.0 nd 0 5.2nd 4.1 nd nd nd p Value 0.42 nd na 0.14 nd 0.21 nd nd nd 95% CI of 0.37nd na 0.59 nd 0.45 nd nd nd OR Quart2 11 nd na 45 nd 37 nd nd nd ORQuart 3 0.50 nd 4.1 2.0 nd 1.0 nd nd nd p Value 0.57 nd 0.21 0.57 nd 1.0nd nd nd 95% CI of 0.044 nd 0.45 0.18 nd 0.062 nd nd nd OR Quart3 5.5 nd37 22 nd 16 nd nd nd OR Quart 4 1.5 nd 2.0 3.0 nd 2.0 nd nd nd p Value0.65 nd 0.57 0.34 nd 0.57 nd nd nd 95% CI of 0.25 nd 0.18 0.31 nd 0.18nd nd nd OR Quart4 9.2 nd 22 29 nd 22 nd nd nd Insulin-like growthfactor-binding protein 7 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 sCr or UO Median 52.8 68.7 52.8 226 52.8 102 Average76.8 280 76.8 332 76.8 151 Stdev 80.0 492 80.0 315 80.0 123 p(t-test)1.4E−13 3.2E−28 0.015 Min 1.00E−9 23.1 1.00E−9 20.4 1.00E−9 33.8 Max 6941830 694 1250 694 382 n (Samp) 933 13 933 16 933 7 n (Patient) 344 13344 16 344 7 sCr only Median 54.0 163 nd nd nd nd Average 84.3 201 nd ndnd nd Stdev 111 203 nd nd nd nd p(t-test) 0.0058 nd nd nd nd Min 1.00E−923.1 nd nd nd nd Max 1830 594 nd nd nd nd n (Samp) 970 7 nd nd nd nd n(Patient) 354 7 nd nd nd nd UO only Median 53.6 114 53.6 283 nd ndAverage 76.5 375 76.5 354 nd nd Stdev 76.0 615 76.0 332 nd nd p(t-test)5.7E−18 2.0E−30 nd nd Min 1.00E−9 46.5 1.00E−9 20.4 nd nd Max 543 1830543 1250 nd nd n (Samp) 800 8 800 14 nd nd n (Patient) 263 8 263 14 ndnd 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKIstage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UOsCr only UO only AUC 0.69 0.70 0.73 0.86 nd 0.85 0.73 nd nd SE 0.0820.11 0.10 0.059 nd 0.065 0.11 nd nd p 0.023 0.069 0.024 1.8E−9 nd 5.3E−80.037 nd nd nCohort 1 933 970 800 933 nd 800 933 nd nd nCohort 2 13 7 816 nd 14 7 nd nd Cutoff 1 49.1 68.6 57.5 118 nd 118 74.9 nd nd Sens 177% 71% 75% 75% nd 71% 71% nd nd Spec 1 47% 61% 54% 82% nd 82% 67% nd ndCutoff 2 46.5 39.0 49.1 104 nd 99.9 51.8 nd nd Sens 2 85% 86% 88% 81% nd86% 86% nd nd Spec 2 45% 35% 46% 79% nd 78% 49% nd nd Cutoff 3 39.0 23.146.5 65.8 nd 65.8 33.8 nd nd Sens 3 92% 100%  100%  94% nd 93% 100%  ndnd Spec 3 36% 19% 44% 61% nd 60% 30% nd nd Cutoff 4 82.1 84.7 82.1 82.1nd 82.1 82.1 nd nd Sens 4 46% 57% 50% 88% nd 86% 57% nd nd Spec 4 70%70% 70% 70% nd 70% 70% nd nd Cutoff 5 110 118 110 110 nd 110 110 nd ndSens 5 46% 57% 50% 75% nd 71% 43% nd nd Spec 5 80% 80% 80% 80% nd 80%80% nd nd Cutoff 6 159 175 159 159 nd 159 159 nd nd Sens 6 46% 43% 50%56% nd 57% 43% nd nd Spec 6 90% 90% 90% 90% nd 90% 90% nd nd OR Quart 23.0 1.0 >2.0 0 nd 0 >2.0 nd nd p Value 0.34 1.0 <0.57 na nd na <0.57 ndnd 95% CI of 0.31 0.062 >0.18 na nd na >0.18 nd nd OR Quart2 29 16 na nand na na nd nd OR Quart 3 3.0 1.0 >2.0 1.0 nd 1.0 >1.0 nd nd p Value0.34 1.0 <0.57 1.0 nd 1.0 <1.00 nd nd 95% CI of 0.31 0.062 >0.18 0.062nd 0.062 >0.062 nd nd OR Quart3 29 16 na 16 nd 16 na nd nd OR Quart 46.1 4.0 >4.1 15 nd 13 >4.1 nd nd p Value 0.095 0.21 <0.21 0.0096 nd0.015 <0.21 nd nd 95% CI of 0.73 0.45 >0.45 1.9 nd 1.6 >0.45 nd nd ORQuart4 51 36 na 110 nd 98 na nd nd

TABLE 10 Comparison of marker levels in EDTA samples collected fromCohort 1 (patients that did not progress beyond RIFLE stage 0, R, or I)and in EDTA samples collected from Cohort 2 (subjects who progress toRIFLE stage F) at 0, 24 hours, and 48 hours prior to the subjectreaching RIFLE stage I. Cancer Antigen 19-9 0 hr prior to AKI stage 24hr prior to AKI stage 48 hr prior to AKI stage sCr or UO Cohort 1 Cohort2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median nd nd 0.244 0.663 nd ndAverage nd nd 0.645 1.43 nd nd Stdev nd nd 2.70 1.89 nd nd p(t-test) ndnd 0.48 nd nd Min nd nd 1.00E−9 0.401 nd nd Max nd nd 41.6 5.26 nd nd n(Samp) nd nd 248 6 nd nd n (Patient) nd nd 158 6 nd nd 0 hr prior to AKIstage 24 hr prior to AKI stage 48 hr prior to AKI stage sCr or UO sCronly UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUCnd nd nd 0.82 nd nd nd nd nd SE nd nd nd 0.11 nd nd nd nd nd p nd nd nd0.0022 nd nd nd nd nd nCohort 1 nd nd nd 248 nd nd nd nd nd nCohort 2 ndnd nd 6 nd nd nd nd nd Cutoff 1 nd nd nd 0.586 nd nd nd nd nd Sens 1 ndnd nd 83% nd nd nd nd nd Spec 1 nd nd nd 77% nd nd nd nd nd Cutoff 2 ndnd nd 0.586 nd nd nd nd nd Sens 2 nd nd nd 83% nd nd nd nd nd Spec 2 ndnd nd 77% nd nd nd nd nd Cutoff 3 nd nd nd 0.374 nd nd nd nd nd Sens 3nd nd nd 100%  nd nd nd nd nd Spec 3 nd nd nd 67% nd nd nd nd nd Cutoff4 nd nd nd 0.452 nd nd nd nd nd Sens 4 nd nd nd 83% nd nd nd nd nd Spec4 nd nd nd 71% nd nd nd nd nd Cutoff 5 nd nd nd 0.633 nd nd nd nd ndSens 5 nd nd nd 50% nd nd nd nd nd Spec 5 nd nd nd 81% nd nd nd nd ndCutoff 6 nd nd nd 1.41 nd nd nd nd nd Sens 6 nd nd nd 17% nd nd nd nd ndSpec 6 nd nd nd 90% nd nd nd nd nd OR Quart 2 nd nd nd >0 nd nd nd nd ndp Value nd nd nd <na   nd nd nd nd nd 95% CI of nd nd nd >na   nd nd ndnd nd OR Quart2 nd nd nd na nd nd nd nd nd OR Quart 3 nd nd nd >1.0 ndnd nd nd nd p Value nd nd nd <0.99 nd nd nd nd nd 95% CI of nd ndnd >0.062 nd nd nd nd nd OR Quart3 nd nd nd na nd nd nd nd nd OR Quart 4nd nd nd >5.3 nd nd nd nd nd p Value nd nd nd <0.13 nd nd nd nd nd 95%CI of nd nd nd >0.61 nd nd nd nd nd OR Quart4 nd nd nd na nd nd nd nd ndC-C motif chemokine 13 0 hr prior to AKI stage 24 hr prior to AKI stage48 hr prior to AKI stage sCr or UO Cohort 1 Cohort 2 Cohort 1 Cohort 2Cohort 1 Cohort 2 Median nd nd 21.0 30.9 nd nd Average nd nd 61.7 29.1nd nd Stdev nd nd 207 27.1 nd nd p(t-test) nd nd 0.70 nd nd Min nd nd1.00E−9 1.00E−9 nd nd Max nd nd 2520 68.2 nd nd n (Samp) nd nd 177 6 ndnd n (Patient) nd nd 126 6 nd nd 0 hr prior to AKI stage 24 hr prior toAKI stage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr or UOsCr only UO only sCr or UO sCr only UO only AUC nd nd nd 0.50 nd nd ndnd nd SE nd nd nd 0.12 nd nd nd nd nd p nd nd nd 0.99 nd nd nd nd ndnCohort 1 nd nd nd 177 nd nd nd nd nd nCohort 2 nd nd nd 6 nd nd nd ndnd Cutoff 1 nd nd nd 0 nd nd nd nd nd Sens 1 nd nd nd 100%  nd nd nd ndnd Spec 1 nd nd nd 0% nd nd nd nd nd Cutoff 2 nd nd nd 0 nd nd nd nd ndSens 2 nd nd nd 100%  nd nd nd nd nd Spec 2 nd nd nd 0% nd nd nd nd ndCutoff 3 nd nd nd 0 nd nd nd nd nd Sens 3 nd nd nd 100%  nd nd nd nd ndSpec 3 nd nd nd 0% nd nd nd nd nd Cutoff 4 nd nd nd 54.3 nd nd nd nd ndSens 4 nd nd nd 17%  nd nd nd nd nd Spec 4 nd nd nd 71%  nd nd nd nd ndCutoff 5 nd nd nd 72.2 nd nd nd nd nd Sens 5 nd nd nd 0% nd nd nd nd ndSpec 5 nd nd nd 81%  nd nd nd nd nd Cutoff 6 nd nd nd 111 nd nd nd nd ndSens 6 nd nd nd 0% nd nd nd nd nd Spec 6 nd nd nd 90%  nd nd nd nd nd ORQuart 2 nd nd nd 2.0 nd nd nd nd nd p Value nd nd nd 0.56 nd nd nd nd nd95% CI of nd nd nd 0.18 nd nd nd nd nd OR Quart2 nd nd nd 23 nd nd nd ndnd OR Quart 3 nd nd nd 3.1 nd nd nd nd nd p Value nd nd nd 0.33 nd nd ndnd nd 95% CI of nd nd nd 0.31 nd nd nd nd nd OR Quart3 nd nd nd 31 nd ndnd nd nd OR Quart 4 nd nd nd 0 nd nd nd nd nd p Value nd nd nd na nd ndnd nd nd 95% CI of nd nd nd na nd nd nd nd nd OR Quart4 nd nd nd na ndnd nd nd nd C-X-C motif chemokine 6 0 hr prior to AKI stage 24 hr priorto AKI stage 48 hr prior to AKI stage sCr or UO Cohort 1 Cohort 2 Cohort1 Cohort 2 Cohort 1 Cohort 2 Median nd nd 28.2 32.4 nd nd Average nd nd45.6 31.0 nd nd Stdev nd nd 50.4 15.9 nd nd p(t-test) nd nd 0.48 nd ndMin nd nd 3.49 9.69 nd nd Max nd nd 311 48.8 nd nd n (Samp) nd nd 178 6nd nd n (Patient) nd nd 127 6 nd nd 0 hr prior to AKI stage 24 hr priorto AKI stage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr orUO sCr only UO only sCr or UO sCr only UO only AUC nd nd nd 0.49 nd ndnd nd nd SE nd nd nd 0.12 nd nd nd nd nd p nd nd nd 0.96 nd nd nd nd ndnCohort 1 nd nd nd 178 nd nd nd nd nd nCohort 2 nd nd nd 6 nd nd nd ndnd Cutoff 1 nd nd nd 17.9 nd nd nd nd nd Sens 1 nd nd nd 83% nd nd nd ndnd Spec 1 nd nd nd 34% nd nd nd nd nd Cutoff 2 nd nd nd 17.9 nd nd nd ndnd Sens 2 nd nd nd 83% nd nd nd nd nd Spec 2 nd nd nd 34% nd nd nd nd ndCutoff 3 nd nd nd 9.55 nd nd nd nd nd Sens 3 nd nd nd 100%  nd nd nd ndnd Spec 3 nd nd nd  9% nd nd nd nd nd Cutoff 4 nd nd nd 45.4 nd nd nd ndnd Sens 4 nd nd nd 17% nd nd nd nd nd Spec 4 nd nd nd 70% nd nd nd nd ndCutoff 5 nd nd nd 63.5 nd nd nd nd nd Sens 5 nd nd nd  0% nd nd nd nd ndSpec 5 nd nd nd 80% nd nd nd nd nd Cutoff 6 nd nd nd 107 nd nd nd nd ndSens 6 nd nd nd  0% nd nd nd nd nd Spec 6 nd nd nd 90% nd nd nd nd nd ORQuart 2 nd nd nd >3.2 nd nd nd nd nd p Value nd nd nd <0.32 nd nd nd ndnd 95% CI of nd nd nd >0.32 nd nd nd nd nd OR Quart2 nd nd nd na nd ndnd nd nd OR Quart 3 nd nd nd >2.1 nd nd nd nd nd p Value nd nd nd <0.55nd nd nd nd nd 95% CI of nd nd nd >0.18 nd nd nd nd nd OR Quart3 nd ndnd na nd nd nd nd nd OR Quart 4 nd nd nd >1.0 nd nd nd nd nd p Value ndnd nd <0.99 nd nd nd nd nd 95% CI of nd nd nd >0.062 nd nd nd nd nd ORQuart4 nd nd nd na nd nd nd nd nd Coagulation factor VII 0 hr prior toAKI stage 24 hr prior to AKI stage 48 hr prior to AKI stage Cohort 1Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 sCr or UO Median 301 274301 191 nd nd Average 321 283 321 218 nd nd Stdev 174 138 174 135 nd ndp(t-test) 0.45 0.052 nd nd Min 2.08 77.8 2.08 32.8 nd nd Max 1020 5491020 418 nd nd n (Samp) 551 12 551 11 nd nd n (Patient) 213 12 213 11 ndnd UO only Median 289 379 289 286 nd nd Average 307 384 307 231 nd ndStdev 165 151 165 138 nd nd p(t-test) 0.19 0.18 nd nd Min 8.62 158 8.6232.8 nd nd Max 1020 611 1020 418 nd nd n (Samp) 465 8 465 9 nd nd n(Patient) 174 8 174 9 nd nd 0 hr prior to AKI stage 24 hr prior to AKIstage 48 hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCronly UO only sCr or UO sCr only UO only AUC 0.45 nd 0.65 0.33 nd 0.38 ndnd nd SE 0.087 nd 0.11 0.091 nd 0.10 nd nd nd p 0.54 nd 0.16 0.067 nd0.23 nd nd nd nCohort 1 551 nd 465 551 nd 465 nd nd nd nCohort 2 12 nd 811 nd 9 nd nd nd Cutoff 1 192 nd 283 101 nd 100 nd nd nd Sens 1 75% nd75% 73% nd 78% nd nd nd Spec 1 28% nd 49%  8% nd  8% nd nd nd Cutoff 2156 nd 261 79.5 nd 79.5 nd nd nd Sens 2 83% nd 88% 82% nd 89% nd nd ndSpec 2 18% nd 45%  5% nd  6% nd nd nd Cutoff 3 130 nd 156 74.8 nd 25.4nd nd nd Sens 3 92% nd 100%  91% nd 100%  nd nd nd Spec 3 12% nd 19%  5%nd  1% nd nd nd Cutoff 4 403 nd 383 403 nd 383 nd nd nd Sens 4 17% nd38%  9% nd 11% nd nd nd Spec 4 70% nd 70% 70% nd 70% nd nd nd Cutoff 5469 nd 448 469 nd 448 nd nd nd Sens 5  8% nd 38%  0% nd  0% nd nd ndSpec 5 80% nd 80% 80% nd 80% nd nd nd Cutoff 6 563 nd 530 563 nd 530 ndnd nd Sens 6  0% nd 25%  0% nd  0% nd nd nd Spec 6 90% nd 90% 90% nd 90%nd nd nd OR Quart 2 1.0 nd 2.0 >5.2 nd 3.1 nd nd nd p Value 1.0 nd 0.57<0.13 nd 0.33 nd nd nd 95% CI of 0.14 nd 0.18 >0.60 nd 0.32 nd nd nd ORQuart2 7.2 nd 23 na nd 30 nd nd nd OR Quart 3 2.6 nd 2.0 >1.0 nd 1.0 ndnd nd p Value 0.27 nd 0.57 <1.00 nd 1.0 nd nd nd 95% CI of 0.49 nd0.18 >0.062 nd 0.062 nd nd nd OR Quart3 13 nd 23 na nd 16 nd nd nd ORQuart 4 1.5 nd 3.0 >5.2 nd 4.1 nd nd nd p Value 0.65 nd 0.34 <0.13 nd0.21 nd nd nd 95% CI of 0.25 nd 0.31 >0.60 nd 0.46 nd nd nd OR Quart49.3 nd 30 na nd 38 nd nd nd nsulin-like growth factor-binding protein 70 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior to AKIstage sCr or UO Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2Median nd nd 69.2 172 nd nd Average nd nd 74.9 194 nd nd Stdev nd nd32.9 137 nd nd p(t-test) nd nd 6.6E−13 nd nd Min nd nd 18.6 53.8 nd ndMax nd nd 250 437 nd nd n (Samp) nd nd 248 6 nd nd n (Patient) nd nd 1586 nd nd 0 hr prior to AKI stage 24 hr prior to AKI stage 48 hr prior toAKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr orUO sCr only UO only AUC nd nd nd 0.84 nd nd nd nd nd SE nd nd nd 0.10 ndnd nd nd nd p nd nd nd 9.7E−4 nd nd nd nd nd nCohort 1 nd nd nd 248 ndnd nd nd nd nCohort 2 nd nd nd 6 nd nd nd nd nd Cutoff 1 nd nd nd 93.0nd nd nd nd nd Sens 1 nd nd nd 83% nd nd nd nd nd Spec 1 nd nd nd 78% ndnd nd nd nd Cutoff 2 nd nd nd 93.0 nd nd nd nd nd Sens 2 nd nd nd 83% ndnd nd nd nd Spec 2 nd nd nd 78% nd nd nd nd nd Cutoff 3 nd nd nd 53.5 ndnd nd nd nd Sens 3 nd nd nd 100%  nd nd nd nd nd Spec 3 nd nd nd 30% ndnd nd nd nd Cutoff 4 nd nd nd 85.5 nd nd nd nd nd Sens 4 nd nd nd 83% ndnd nd nd nd Spec 4 nd nd nd 70% nd nd nd nd nd Cutoff 5 nd nd nd 96.3 ndnd nd nd nd Sens 5 nd nd nd 67% nd nd nd nd nd Spec 5 nd nd nd 80% nd ndnd nd nd Cutoff 6 nd nd nd 116 nd nd nd nd nd Sens 6 nd nd nd 67% nd ndnd nd nd Spec 6 nd nd nd 90% nd nd nd nd nd OR Quart 2 nd nd nd >1.0 ndnd nd nd nd p Value nd nd nd <1.0 nd nd nd nd nd 95% CI of nd ndnd >0.061 nd nd nd nd nd OR Quart2 nd nd nd na nd nd nd nd nd OR Quart 3nd nd nd >0 nd nd nd nd nd p Value nd nd nd <na   nd nd nd nd nd 95% CIof nd nd nd >na   nd nd nd nd nd OR Quart3 nd nd nd na nd nd nd nd nd ORQuart 4 nd nd nd >5.3 nd nd nd nd nd p Value nd nd nd <0.13 nd nd nd ndnd 95% CI of nd nd nd >0.61 nd nd nd nd nd OR Quart4 nd nd nd na nd ndnd nd nd

TABLE 11 Comparison of marker levels in enroll urine samples collectedfrom Cohort 1 (patients that did not progress beyond RIFLE stage 0 or Rwithin 48 hrs) and in enroll urine samples collected from Cohort 2(subjects reaching RIFLE stage I or F within 48 hrs). Enroll samplesfrom patients already at RIFLE stage I or F were included in Cohort 2.Cancer Antigen 19-9 sCr or UO sCr only UO only Cohort 1 Cohort 2 Cohort1 Cohort 2 Cohort 1 Cohort 2 Median 26.6 42.2 nd nd 27.4 51.5 Average107 282 nd nd 106 305 Stdev 259 668 nd nd 256 694 p(t-test) 0.036 nd nd0.030 Min 1.00E−9 1.00E−9 nd nd 1.00E−9 1.00E−9 Max 2000 2940 nd nd 20002940 n (Samp) 109 24 nd nd 90 22 n (Patient) 109 24 nd nd 90 22 AtEnrollment sCr or UO sCr only UO only AUC 0.60 nd 0.60 SE 0.066 nd 0.070p 0.15 nd 0.13 nCohort 1 109 nd 90 nCohort 2 24 nd 22 Cutoff 1 23.0 nd23.0 Sens 1 71% nd 73% Spec 1 46% nd 42% Cutoff 2 9.92 nd 18.7 Sens 283% nd 82% Spec 2 27% nd 39% Cutoff 3 5.76 nd 9.72 Sens 3 92% nd 91%Spec 3 20% nd 26% Cutoff 4 63.8 nd 57.3 Sens 4 33% nd 45% Spec 4 71% nd70% Cutoff 5 117 nd 114 Sens 5 33% nd 36% Spec 5 81% nd 80% Cutoff 6 240nd 240 Sens 6 17% nd 18% Spec 6 91% nd 90% OR Quart 2 1.3 nd 1.3 p Value0.72 nd 0.72 95% CI of 0.31 nd 0.31 OR Quart2 5.3 nd 5.5 OR Quart 3 2.0nd 1.3 p Value 0.33 nd 0.72 95% CI of 0.51 nd 0.31 OR Quart3 7.4 nd 5.5OR Quart 4 2.2 nd 2.4 p Value 0.23 nd 0.20 95% CI of 0.60 nd 0.63 ORQuart4 8.3 nd 9.2 C-X-C motif chemokine 6 sCr or UO sCr only UO onlyCohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 13.3 33.115.0 42.2 14.9 33.4 Average 27.7 104 38.1 120 28.4 111 Stdev 53.4 18890.0 195 51.5 197 p(t-test) 6.4E−9 0.0036 8.9E−8 Min 1.00E−9 1.621.00E−9 1.62 1.00E−9 3.96 Max 561 909 909 698 561 909 n (Samp) 296 61342 12 217 55 n (Patient) 296 61 342 12 217 55 At Enrollment sCr or UOsCr only UO only AUC 0.71 0.70 0.71 SE 0.039 0.086 0.042 p 7.0E−8 0.0178.0E−7 nCohort 1 296 342 217 nCohort 2 61 12 55 Cutoff 1 17.2 18.8 17.2Sens 1 70% 75% 71% Spec 1 58% 58% 55% Cutoff 2 11.5 11.5 12.3 Sens 2 80%83% 80% Spec 2 45% 41% 44% Cutoff 3 8.44 10.6 8.22 Sens 3 90% 92% 91%Spec 3 38% 40% 34% Cutoff 4 26.0 28.7 27.4 Sens 4 57% 58% 58% Spec 4 70%70% 70% Cutoff 5 37.4 42.2 37.4 Sens 5 48% 50% 49% Spec 5 80% 80% 80%Cutoff 6 53.8 62.4 53.8 Sens 6 33% 42% 33% Spec 6 90% 90% 90% OR Quart 21.8 2.0 2.4 p Value 0.30 0.57 0.12 95% CI of 0.61 0.18 0.80 OR Quart25.0 22 7.4 OR Quart 3 3.0 3.1 3.0 p Value 0.028 0.34 0.050 95% CI of 1.10.31 1.00 OR Quart3 8.2 30 8.9 OR Quart 4 6.6 6.3 7.8 p Value 8.5E−50.092 9.8E−5 95% CI of 2.6 0.74 2.8 OR Quart4 17 53 22 Coagulationfactor VII sCr or UO sCr only UO only Cohort 1 Cohort 2 Cohort 1 Cohort2 Cohort 1 Cohort 2 Median 3.72 7.55 4.03 13.2 3.86 7.85 Average 6.3411.8 7.00 16.5 6.29 10.9 Stdev 7.90 11.2 8.33 15.3 7.90 9.42 p(t-test)0.0011 0.0029 0.0069 Min 0.00408 0.348 0.00408 0.348 0.00408 0.682 Max48.9 47.7 48.9 47.7 48.9 36.7 n (Samp) 139 35 165 8 112 30 n (Patient)139 35 165 8 112 30 At Enrollment sCr or UO sCr only UO only AUC 0.670.72 0.67 SE 0.054 0.10 0.059 p 0.0017 0.038 0.0031 nCohort 1 139 165112 nCohort 2 35 8 30 Cutoff 1 4.79 6.05 4.79 Sens 1 71% 75% 70% Spec 157% 65% 57% Cutoff 2 2.33 5.29 2.33 Sens 2 80% 88% 80% Spec 2 40% 58%40% Cutoff 3 1.01 0.313 1.24 Sens 3 91% 100%  90% Spec 3 18%  3% 23%Cutoff 4 7.01 7.52 7.27 Sens 4 54% 50% 57% Spec 4 71% 70% 71% Cutoff 59.02 10.2 8.49 Sens 5 43% 50% 43% Spec 5 81% 80% 80% Cutoff 6 15.7 18.714.3 Sens 6 31% 50% 33% Spec 6 91% 90% 90% OR Quart 2 0.97 0 0.75 pValue 0.97 na 0.69 95% CI of 0.26 na 0.18 OR Quart2 3.6 na 3.1 OR Quart3 2.3 3.1 1.8 p Value 0.16 0.33 0.36 95% CI of 0.71 0.31 0.52 OR Quart37.4 32 6.1 OR Quart 4 3.9 4.2 3.4 p Value 0.017 0.21 0.040 95% CI of 1.30.45 1.1 OR Quart4 12 39 11 Insulin-like growth factor-binding protein 7sCr or UO sCr only UO only Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1Cohort 2 Median 51.1 131 57.2 306 54.4 131 Average 73.2 213 90.2 29576.6 209 Stdev 75.7 221 115 240 78.9 217 p(t-test) 1.7E−16 1.7E−83.1E−12 Min 1.06 20.4 1.06 23.1 1.06 20.4 Max 533 1250 1250 694 533 1250n (Samp) 294 61 340 12 215 55 n (Patient) 294 61 340 12 215 55 AtEnrollment sCr or UO sCr only UO only AUC 0.77 0.76 0.76 SE 0.037 0.0820.040 p 1.3E−12 0.0015 3.9E−11 nCohort 1 294 340 215 nCohort 2 61 12 55Cutoff 1 73.6 88.9 73.6 Sens 1 70% 75% 71% Spec 1 67% 69% 64% Cutoff 255.8 39.0 61.3 Sens 2 80% 83% 80% Spec 2 55% 35% 55% Cutoff 3 38.2 23.138.4 Sens 3 90% 92% 91% Spec 3 38% 20% 38% Cutoff 4 82.8 90.1 85.1 Sens4 67% 67% 65% Spec 4 70% 70% 70% Cutoff 5 102 120 104 Sens 5 59% 67% 58%Spec 5 80% 80% 80% Cutoff 6 150 199 146 Sens 6 41% 58% 44% Spec 6 90%90% 90% OR Quart 2 1.6 0.49 2.4 p Value 0.40 0.57 0.16 95% CI of 0.510.044 0.70 OR Quart2 5.2 5.6 8.2 OR Quart 3 3.1 0.49 3.8 p Value 0.0380.57 0.027 95% CI of 1.1 0.044 1.2 OR Quart3 9.0 5.6 12 OR Quart 4 104.3 12 p Value 4.9E−6  0.070 1.6E−5  95% CI of 3.8 0.89 3.8 OR Quart4 2821 36

TABLE 12 Comparison of marker levels in enroll EDTA samples collectedfrom Cohort 1 (patients that did not progress beyond RIFLE stage 0 or Rwithin 48 hrs) and in enroll EDTA samples collected from Cohort 2(subjects reaching RIFLE stage I or F within 48 hrs). Enroll samplesfrom patients already at stage I or F were included in Cohort 2. CancerAntigen 19-9 sCr or UO sCr only UO only Cohort 1 Cohort 2 Cohort 1Cohort 2 Cohort 1 Cohort 2 Median 0.228 0.552 nd nd 0.228 0.452 Average0.462 0.989 nd nd 0.503 0.942 Stdev 0.734 1.34 nd nd 0.784 1.41p(t-test) 0.020 nd nd 0.089 Min 1.00E−9 0.0291 nd nd 1.00E−9 0.0291 Max5.30 5.26 nd nd 5.30 5.26 n (Samp) 78 20 nd nd 67 17 n (Patient) 78 20nd nd 67 17 At Enrollment sCr or UO sCr only UO only AUC 0.70 nd 0.65 SE0.071 nd 0.079 p 0.0042 nd 0.054 nCohort 1 78 nd 67 nCohort 2 20 nd 17Cutoff 1 0.332 nd 0.259 Sens 1 70% nd 71% Spec 1 68% nd 54% Cutoff 20.257 nd 0.228 Sens 2 80% nd 82% Spec 2 56% nd 51% Cutoff 3 0.147 nd0.134 Sens 3 90% nd 94% Spec 3 32% nd 28% Cutoff 4 0.361 nd 0.523 Sens 465% nd 47% Spec 4 71% nd 70% Cutoff 5 0.633 nd 0.652 Sens 5 35% nd 29%Spec 5 81% nd 81% Cutoff 6 1.34 nd 1.52 Sens 6 15% nd 12% Spec 6 91% nd91% OR Quart 2 4.4 nd 1.6 p Value 0.20 nd 0.64 95% CI of 0.45 nd 0.24 ORQuart2 42 nd 11 OR Quart 3 12 nd 4.8 p Value 0.028 nd 0.075 95% CI of1.3 nd 0.85 OR Quart3 100 nd 26 OR Quart 4 8.9 nd 3.0 p Value 0.049 nd0.23 95% CI of 1.0 nd 0.51 OR Quart4 79 nd 17 C-X-C motif chemokine 6sCr or UO sCr only UO only Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1Cohort 2 Median 23.9 29.6 24.0 42.6 21.8 28.8 Average 38.1 38.9 37.051.3 38.1 34.3 Stdev 49.2 32.2 45.9 43.2 52.2 20.1 p(t-test) 0.95 0.430.79 Min 4.02 9.69 4.02 9.69 4.02 12.3 Max 311 144 311 144 311 88.5 n(Samp) 58 18 69 7 50 14 n (Patient) 58 18 69 7 50 14 At Enrollment sCror UO sCr only UO only AUC 0.57 0.67 0.59 SE 0.079 0.12 0.089 p 0.350.14 0.30 nCohort 1 58 69 50 nCohort 2 18 7 14 Cutoff 1 23.3 39.7 23.3Sens 1 72% 71% 71% Spec 1 48% 74% 52% Cutoff 2 16.7 26.0 16.7 Sens 2 83%86% 86% Spec 2 33% 59% 36% Cutoff 3 10.5 8.88 15.8 Sens 3 94% 100%  93%Spec 3 17%  7% 32% Cutoff 4 38.9 38.9 38.7 Sens 4 44% 71% 43% Spec 4 71%71% 70% Cutoff 5 46.0 45.4 46.0 Sens 5 17% 29% 14% Spec 5 81% 81% 80%Cutoff 6 66.6 66.6 61.7 Sens 6 11% 14%  7% Spec 6 91% 91% 90% OR Quart 21.4 0 5.0 p Value 0.68 na 0.17 95% CI of 0.27 na 0.49 OR Quart2 7.4 na51 OR Quart 3 3.1 4.8 9.0 p Value 0.15 0.18 0.057 95% CI of 0.66 0.480.94 OR Quart3 15 48 87 OR Quart 4 1.4 2.1 3.5 p Value 0.68 0.55 0.3195% CI of 0.27 0.18 0.32 OR Quart4 7.4 26 37 Coagulation factor VII sCror UO sCr only UO only Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1Cohort 2 Median 287 225 280 173 276 268 Average 320 239 308 201 298 250Stdev 182 142 179 90.2 159 149 p(t-test) 0.014 0.077 0.14 Min 2.08 12.22.08 75.6 35.7 12.2 Max 950 571 950 380 809 571 n (Samp) 140 36 166 9112 31 n (Patient) 140 36 166 9 112 31 At Enrollment sCr or UO sCr onlyUO only AUC 0.37 0.32 0.42 SE 0.055 0.10 0.060 p 0.020 0.069 0.18nCohort 1 140 166 112 nCohort 2 36 9 31 Cutoff 1 153 158 153 Sens 1 72%78% 71% Spec 1 19% 22% 21% Cutoff 2 102 129 102 Sens 2 81% 89% 81% Spec2  9% 14% 10% Cutoff 3 64.8 74.8 64.8 Sens 3 92% 100%  90% Spec 3  4% 7% 3 % Cutoff 4 376 368 361 Sens 4 19% 11% 23% Spec 4 71% 71% 71%Cutoff 5 463 458 456 Sens 5  6%  0%  6% Spec 5 80% 80% 80% Cutoff 6 574537 526 Sens 6  0%  0%  6% Spec 6 90% 90% 90% OR Quart 2 1.4 >2.1 1.2 pValue 0.56 <0.55 0.77 95% CI of 0.44 >0.18 0.38 OR Quart2 4.5 na 3.7 ORQuart 3 1.4 >3.2 0.67 p Value 0.56 <0.32 0.53 95% CI of 0.44 >0.32 0.19OR Quart3 4.5 na 2.3 OR Quart 4 3.0 >4.5 1.9 p Value 0.047 <0.19 0.2595% CI of 1.0 >0.48 0.64 OR Quart4 8.6 na 5.7 Insulin-like growthfactor-binding protein 7 sCr or UO sCr only UO only Cohort 1 Cohort 2Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 73.2 97.8 nd nd 73.5 96.3Average 75.2 124 nd nd 78.8 120 Stdev 31.9 95.9 nd nd 32.8 100.0p(t-test) 2.3E−4 nd nd 0.0049 Min 18.6 43.7 nd nd 38.1 43.7 Max 188 437nd nd 188 437 n (Samp) 78 20 nd nd 67 17 n (Patient) 78 20 nd nd 67 17At Enrollment sCr or UO sCr only UO only AUC 0.68 nd 0.63 SE 0.072 nd0.079 p 0.014 nd 0.11 nCohort 1 78 nd 67 nCohort 2 20 nd 17 Cutoff 166.7 nd 57.2 Sens 1 70% nd 71% Spec 1 46% nd 34% Cutoff 2 55.2 nd 53.3Sens 2 80% nd 82% Spec 2 33% nd 28% Cutoff 3 52.0 nd 49.8 Sens 3 90% nd94% Spec 3 23% nd 15% Cutoff 4 84.3 nd 86.1 Sens 4 60% nd 59% Spec 4 71%nd 70% Cutoff 5 91.7 nd 97.1 Sens 5 60% nd 47% Spec 5 81% nd 81% Cutoff6 114 nd 128 Sens 6 40% nd 24% Spec 6 91% nd 91% OR Quart 2 1.8 nd 1.4 pValue 0.48 nd 0.68 95% CI of 0.37 nd 0.27 OR Quart2 8.3 nd 7.3 OR Quart3 0.30 nd 0.63 p Value 0.32 nd 0.64 95% CI of 0.029 nd 0.094 OR Quart33.2 nd 4.2 OR Quart 4 5.5 nd 3.7 p Value 0.021 nd 0.089 95% CI of 1.3 nd0.82 OR Quart4 23 nd 17

Example 7 Kidney Injury Markers for Evaluating Mortality Risk inPatients

Patients from the intensive care unit (ICU) were enrolled in thefollowing study. Each patient was classified by kidney status asnon-injury (0), risk of injury (R), injury (I), and failure (F)according to the maximum stage reached within 48 hours of enrollment asdetermined by the RIFLE criteria. EDTA anti-coagulated blood samples (10mL) and a urine samples (25-30 mL) were collected from each patient atthe time of enrollment into the study. Markers were each measured bystandard immunoassay methods using commercially available assay reagentsin the urine samples and the plasma component of the blood samplescollected.

The patient population was segregated based on the marker concentrationsusing threshold values which divided the population into thirds(“tertiles”). Patients with marker concentrations in the lower, middle,and upper third comprise the first, second, and third tertiles,respectively. The relative risk of AKI-related mortality within 7, 14,and 28 days was calculated for the second and third tertiles, relativeto a value of 1 for the first tertile, as indicated in the followingtable. “AKI-related mortality” or “AKI-related death” was defined asdeath accompanied by a minimum RIFLE stage of R.

TABLE 13 Relative risk of AKI-related death within 7, 14, and 28 daysfrom enrollment for the third tertile compared to the first tertile ofmarker concentrations. Relative Risk Total Total Number for Third Numberof of AKI- Marker Tertile p Patients related Deaths AKI-Related DeathInsulin-like growth factor- 5.5 0.02 355 16 within 7 Days after bindingprotein 7 enrollment C-X-C motif chemokine 6 3.0 0.09 356 16 AKI-RelatedDeath Insulin-like growth factor- 6.5 0.01 355 20 within 14 Days afterbinding protein 7 enrollment C-X-C motif chemokine 6 4.0 0.03 356 20AKI-Related Death Insulin-like growth factor- 4.7 0.01 355 22 within 28Days after binding protein 7 enrollment C-X-C motif chemokine 6 3.0 0.05356 22

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

We claim:
 1. A method of treating a subject comprising: (a) detecting anelevated level of insulin-like growth factor-binding protein 7 (IGFBP7)in a urine sample obtained from the subject as compared to apredetermined threshold level, wherein the threshold level is selectedbased on results of a population study of individuals, and wherein theresults separate the population into a first subpopulation of theindividuals with a level of IGFBP7 that is greater than the thresholdlevel and a second subpopulation of the individuals with a level ofIGFBP7 that is less than the threshold level; (b) determining that thesubject is at risk of a future acute kidney injury within 48 hours ofthe time the urine sample is obtained from the subject based on theelevated level of insulin-like growth factor-binding protein 7 in thesample, and; (c) treating the subject determined to be at risk of thefuture acute kidney injury, wherein the treatment comprises initiatingrenal replacement therapy, ceasing administration of compounds to thesubject that are known to be damaging to the kidney, or modifyingdiuretic administration of compounds, wherein the subject: (i) isundergoing or has undergone an acute medical event which predisposes thesubject for developing the acute kidney injury; or (ii) has been exposedto an agent that predisposes the subject for developing the acute kidneyinjury.
 2. The method of claim 1, wherein the method further comprisescontacting all or a portion of the urine sample with a binding reagentwhich specifically binds to insulin-like growth factor-binding protein7, and generating an assay result indicative of binding of insulin-likegrowth factor-binding protein 7 to the binding reagent.
 3. The method ofclaim 2, wherein the assay result comprises a measured concentration ofinsulin-like growth factor-binding protein
 7. 4. The method of claim 2,wherein the binding reagent is an antibody.
 5. The method of claim 1,wherein the future acute kidney injury is a future RIFLE stage I acutekidney injury or a future RIFLE stage F acute kidney injury.
 6. Themethod of claim 1, wherein the determining further comprises determiningthat the subject has an increased likelihood of a future acute kidneyinjury within 24 hours of the time the urine sample is obtained from thesubject.
 7. The method of claim 1, comprising initiating renalreplacement therapy.
 8. The method of claim 1, comprising ceasingadministration of compounds to the subject that are damaging to thekidney.
 9. The method of claim 1, comprising modifying diureticadministration of compounds used for treatment of a kidney disorder. 10.The method of claim 1, wherein the future acute kidney injury isprerenal, intrinsic renal, or postrenal acute renal failure (ARF). 11.The method of claim 1, wherein the subject is not in RIFLE stage I acutekidney injury or RIFLE stage F acute kidney injury.
 12. The method ofclaim 1, wherein the subject is in RIFLE stage
 0. 13. The method ofclaim 1, wherein the subject is in RIFLE stage R.
 14. The method ofclaim 1, wherein the subject is in RIFLE stage I.
 15. The method ofclaim 1, wherein the subject has been diagnosed with one or more ofcongestive heart failure, preeclampsia, eclampsia, diabetes mellitus,hypertension, coronary artery disease, proteinuria, renal insufficiency,glomerular filtration below the normal range, cirrhosis, serumcreatinine above the normal range, and sepsis.
 16. The method of claim1, wherein the subject: (i) is undergoing or has undergone majorvascular surgery, coronary artery bypass, or other cardiac surgery; or(ii) has been exposed to NSAIDs, cyclosporines, tacrolimus,aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin,ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, orstreptozotocin.